Human cytomegalovirus rna vaccines

ABSTRACT

The disclosure describes HCMV ribonucleic acid (RNA) vaccines, as well as methods of using the vaccines and compositions comprising the vaccines.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/006,526, filed Jun. 12, 2018, which is a continuation of U.S.application Ser. No. 15/674,569, filed Aug. 11, 2017, which is acontinuation of international application number PCT/US2016/058310,filed Oct. 21, 2016, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” which waspublished under PCT Article 21(2) in English and which claims thebenefit under 35 U.S.C. § 119(e) of U.S. provisional application No.62/245,166, filed Oct. 22, 2015, entitled “HUMAN CYTOMEGALOVIRUSVACCINE,” U.S. provisional application No. 62/247,614, filed Oct. 28,2015, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” and U.S. provisionalapplication No. 62/245,031, filed Oct. 22, 2015, each of which is hereinincorporated by reference in its entirety.

BACKGROUND

Human cytomegalovirus (HCMV) is a genus of viruses in the orderHerpesvirales, in the family Herpesviridae, in the subfamilyBetaherpesvirinae. There are currently eight species in this genus,which have been identified and classified for different mammals,including humans, monkeys, and rodents. The most studied genus is humancytomegalovirus, also known as human herpesvirus 5 (HHV-5), which iswidely distributed in the human population. Diseases associated withHHV-5 include mononucleosis and pneumonias. All herpesviruses share acharacteristic ability to remain latent within the body over longperiods of time. Although they may be found throughout the body, CMVinfections are frequently associated with the salivary glands in humansand other mammals. Other CMV viruses are found in several mammalspecies, but species isolated from animals differ from HCMV in terms ofgenomic structure, and have not been reported to cause human disease.

HCMV is endemic in most parts of the world. It is a ubiquitous largeenveloped virus that infects 50 to 100% of the adult populationworldwide. Although generally asymptomatic in immunocompetent hosts,HCMV infection is a major cause of morbidity and mortality inimmunocompromised persons, such as infants following congenital orneonatal infections, transplant recipients, or AIDS patients.

Primary infection normally results in subclinical disease after whichthe virus becomes latent, retaining the capacity to reactivate at alater time. The virus is transmitted through body fluids, such as blood,saliva, urine, semen and breast milk. In particular, individuals withundeveloped or compromised immunity are highly sensitive to infection byHCMV. It is estimated that at least 60% of the US population has beenexposed to CMV, with a prevalence of more than 90% in high-risk groups(e.g., unborn babies whose mothers become infected with CMV during thepregnancy or people with HIV).

In healthy individuals, HCMV typically causes an asymptomatic infectionor produces mild, flulike symptoms. However, among two populations, HCMVis responsible for serious medical conditions. First, HCMV is a majorcause of congenital defects in newborns infected in utero. Amongcongenitally infected newborns, 5-10% have major clinical symptoms atbirth, such as microcephaly, intracranial calcifications, and hepatitis,as well as cytomegalic inclusion disease, which affects many tissues andorgans including the central nervous system, liver, and retina and canlead to multi-organ failure and death. Other infants may be asymptomaticat birth, but later develop hearing loss or central nervous systemabnormalities causing, in particular, poor intellectual performance andmental retardation. These pathologies are due in part to the ability ofHCMV to enter and replicate in diverse cell types including epithelialcells, endothelial cells, smooth muscle cells, fibroblasts, neurons, andmonocytes/macrophages.

The second population at risk are immunocompromised patients, such asthose suffering from HIV infection and those undergoingtransplantations. In this situation, the virus becomes an opportunisticpathogen and causes severe disease with high morbidity and mortality.The clinical disease causes a variety of symptoms including fever,pneumonia, hepatitis, encephalitis, myelitis, colitis, uveitis,retinitis, and neuropathy. Rarer manifestations of HCMV infections inimmunocompetent individuals include Guillain-Barré syndrome,meningoencephalitis, pericarditis, myocarditis, thrombocytopenia, andhemolytic anemia. Moreover, HCMV infection increases the risk of organgraft loss through transplant vascular sclerosis and restenosis, and mayincrease atherosclerosis in transplant patients as well as in thegeneral population. It is estimated that HCMV infection causes clinicaldisease in 75% of patients in the first year after transplantation.

There is currently no approved HCMV vaccine. Two candidate vaccines,Towne and gB/MF59, have completed phase II efficacy trials. The Townevaccine appears protective against both infection and disease caused bychallenge with pathogenic Toledo strain and also appears to be effectivein preventing severe post-transplantation CMV disease. However, in asmall phase II clinical trial, a low dose of Towne vaccine failed toshow protection against infection of seronegative mothers who hadchildren actively shedding CMV.

The gB/MF59 vaccine is a protein subunit vaccine comprised of atransmembrane-deleted version of HCMV gB protein, which induces highlevels of fibroblast entry neutralizing antibodies in humans and hasbeen shown to be safe and well tolerated in both adults and toddlers. Arecent phase II double-blind placebo-controlled trial of the gB/MF59vaccine revealed a 50% efficacy in inducing sterilizing immunity. Asthis vaccine induces potent antibody responses but very weak T-cellresponses, the partial efficacy provided by the vaccine is thought to beprimarily antibody-mediated. While this HCMV vaccine is the first toshow any protective efficacy, its 50% protection falls short of the80-90% desired for most vaccines.

In addition, antibody therapy has been used to control HCMV infection inimmunocompromised individuals and to reduce the pathologicalconsequences of maternal-fetal transmission, although such therapy isusually not sufficient to eradicate the virus. HCMV immunoglobulins(Igs) have been administered to transplant patients in association withimmunosuppressive treatments for prophylaxis of HCMV disease with mixedresults. Antibody therapy has also been used to control congenitalinfection and prevent disease in newborns. However, these products areplasma derivatives with relatively low potency and have to beadministered by intravenous infusion at very high doses in order todeliver sufficient amounts of neutralizing antibodies.

HCMV is the leading viral cause of neurodevelopmental abnormality andother birth defects in children and the costs to society aresubstantial. Although antiviral therapy is available, the treatment withantiviral agents is imperfect and development of a CMV vaccine is themost promising strategy for preventing CMV infection. Given that thehealth and economic benefits of effective HCMV vaccines are significant,the US Institute of Medicine and US National Vaccine Program Office hascategorized development of a CMV vaccine as a highest priority, but nocandidate vaccine is under consideration for licensure.

SUMMARY

In view of the lack of HCMV vaccines, there is a significant need for avaccine that would be safe and effective in all patient populations toprevent and/or to treat HCMV infection. In particular, there is a needfor a vaccine that would be safe and effective for immunocompromised,at-risk pregnant women, and infant patients to prevent or to reduce theseverity and/or duration of HCMV. Provided herein is a ribonucleic acid(RNA) vaccine that builds on the knowledge that RNA (e.g., messenger RNA(mRNA)) can safely direct the body's cellular machinery to producenearly any protein of interest, from native proteins to antibodies andother entirely novel protein constructs that can have therapeuticactivity inside and outside of cells. The HCMV RNA vaccines of thepresent disclosure may be used to induce a balanced immune responseagainst human cytomegalovirus comprising both cellular and humoralimmunity, without many of the risks associated with DNA or attenuatedvirus vaccination.

The RNA vaccines may be utilized in various settings depending on theprevalence of the infection or the degree or level of unmet medicalneed. The RNA vaccines may be utilized to treat and/or prevent a HCMV ofvarious genotypes, strains, and isolates. The RNA vaccines have superiorproperties in that they produce much larger antibody titers and produceresponses earlier than commercially available anti-viral therapeutictreatments. While not wishing to be bound by theory, it is believed thatthe RNA vaccines, as mRNA polynucleotides, are better designed toproduce the appropriate protein conformation upon translation as the RNAvaccines co-opt natural cellular machinery. Unlike traditional vaccineswhich are manufactured ex vivo and may trigger unwanted cellularresponses, the RNA vaccines are presented to the cellular system in amore native fashion.

Various human cytomegalovirus amino acid sequences encompasses by thepresent disclosure are provided in Tables 1, 2 and 6 below. RNA vaccinesas provided herein may include at least one RNA polynucleotide encodingat least one of the HCMV proteins provided in Tables 1, 2 or 6, or afragment, homolog (e.g., having at least 80%, 85%, 90%, 95%, 98% or 99%identity) or derivative thereof.

Some embodiments of the present disclosure provide HCMV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment or epitope thereof. Some embodiments of thepresent disclosure provide HCMV vaccines that include at least one RNApolynucleotide having an open reading frame encoding two or more HCMVantigenic polypeptides or an immunogenic fragment or epitope thereof.Some embodiments of the present disclosure provide HCMV vaccines thatinclude two or more RNA polynucleotides having an open reading frameencoding two or more HCMV antigenic polypeptides or immunogenicfragments or epitopes thereof. The one or more HCMV antigenicpolypeptides may be encoded on a single RNA polynucleotide or may beencoded individually on multiple (e.g., two or more) RNApolynucleotides.

In some embodiments, an antigenic polypeptide is an HCMV glycoprotein.For example, a HCMV glycoprotein may be selected from HCMV gH, gL, gB,gO, gN, and gM and an immunogenic fragment or epitope thereof. In someembodiments, the antigenic polypeptide is a HCMV gH polypeptide. In someembodiments, the antigenic polypeptide is a HCMV gL polypeptide. In someembodiments, the antigenic polypeptide is a HCMV gB polypeptide. In someembodiments, the antigenic polypeptide is a HCMV gO polypeptide. In someembodiments, the antigenic polypeptide is a HCMV gN polypeptide. In someembodiments, the antigenic polypeptide is a HCMV gM polypeptide. In someembodiments, the HCMV glycoprotein is encoded by a nucleic acid sequenceof SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO:6, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQID NO:66, SEQ ID NO:68, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:111, SEQ ID NO:112, or SEQ ID NO:113.

In some embodiments, the HCMV glycoprotein is a variant gH polypeptide,a variant gL polypeptide, or a variant gB polypeptide. In someembodiments, the variant HCMV gH, gL, or gB polypeptide is a truncatedpolypeptide lacking one or more of the following domain sequences: (1)the hydrophobic membrane proximal domain, (2) the transmembrane domain,and (3) the cytoplasmic domain. In some embodiments, the truncated HCMVgH, gL, or gB polypeptide lacks the hydrophobic membrane proximaldomain, the transmembrane domain, and the cytoplasmic domain. In someembodiments, the truncated HCMV gH, gL, or gB polypeptide comprises onlythe ectodomain sequence. In some embodiments, the HCMV truncatedglycoprotein is encoded by a nucleic acid sequence of SEQ ID NO: 7, SEQID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:12.

In some embodiments, an antigenic polypeptide is an HCMV proteinselected from UL83, UL123, UL128, UL130 and UL131A or an immunogenicfragment or epitope thereof. In some embodiments, the antigenicpolypeptide is a HCMV UL83 polypeptide. In some embodiments, theantigenic polypeptide is a HCMV UL123 polypeptide. In some embodiments,the antigenic polypeptide is a HCMV UL128 polypeptide. In someembodiments, the antigenic polypeptide is a HCMV UL130 polypeptide. Insome embodiments, the antigenic polypeptide is a HCMV UL131Apolypeptide. In some embodiments, the HCMV protein is encoded by anucleic acid sequence of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, or SEQ ID NO:18.

In some embodiments, the antigenic polypeptide comprises two or moreHCMV proteins, fragments, or epitopes thereof. In some embodiments, theantigenic polypeptide comprises two or more glycoproteins, fragments, orepitopes thereof. In some embodiments, the antigenic polypeptidecomprises at least one HCMV glycoprotein, fragment or epitope thereofand at least one other HCMV protein, fragment or epitope thereof. Insome embodiments, the two or more HCMV polypeptides are encoded by asingle RNA polynucleotide. In some embodiments, the two or more HCMVpolypeptides are encoded by two or more RNA polynucleotides, forexample, each HCMV polypeptide is encoded by a separate RNApolynucleotide. In some embodiments, the two or more HCMV glycoproteinscan be any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptidesor immunogenic fragments or epitopes thereof. In some embodiments, thetwo or more glycoproteins can be any combination of HCMV gB and one ormore HCMV polypeptides selected from gH, gL, gO, gN, and gM polypeptidesor immunogenic fragments or epitopes thereof. In some embodiments, thetwo or more glycoproteins can be any combination of HCMV gH and one ormore HCMV polypeptides selected from gL, gO, gN, and gM polypeptides orimmunogenic fragments or epitopes thereof. In some embodiments, the twoor more glycoproteins can be any combination of HCMV gL and one or moreHCMV polypeptides selected from gB, gH, gO, gN, and gM polypeptides orimmunogenic fragments or epitopes thereof. In some embodiments, the twoor more HCMV glycoproteins are gB and gH. In some embodiments, the twoor more HCMV glycoproteins are gB and gL. In some embodiments, the twoor more HCMV glycoproteins are gH and gL. In some embodiments, the twoor more HCMV glycoproteins are gB, gL, and gH. In some embodiments, thetwo or more HCMV proteins can be any combination of HCMV UL83, UL123,UL128, UL130, and UL131A polypeptides or immunogenic fragments orepitopes thereof. In some embodiments, the two or more HCMVglycoproteins are UL123 and UL130. In some embodiments, the two or moreHCMV glycoproteins are UL123 and 131A. In some embodiments, the two ormore HCMV glycoproteins are UL130 and 131A. In some embodiments, the twoor more HCMV glycoproteins are UL 128, UL130 and 131A. In someembodiments, the two or more HCMV proteins can be any combination ofHCMV gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, and UL131Apolypeptides or immunogenic fragments or epitopes thereof. In someembodiments, the two or more glycoproteins can be any combination ofHCMV gH and one or more HCMV polypeptides selected from gL, UL128,UL130, and UL131A polypeptides or immunogenic fragments or epitopesthereof. In some embodiments, the two or more glycoproteins can be anycombination of HCMV gL and one or more HCMV polypeptides selected fromgH, UL128, UL130, and UL131A polypeptides or immunogenic fragments orepitopes thereof. In some embodiments, the two or more HCMVglycoproteins are gL, gH, UL 128, UL130 and 131A. In any of theseembodiments in which the vaccine comprises two or more HCMV proteins,the HCMV gH may be a variant gH, such as any of the variant HCMV gHglycoproteins disclosed herein, for example, any of the variant HCMV gHdisclosed in the preceding paragraphs and in the Examples. In any ofthese embodiments in which the vaccine comprises two or more HCMVproteins, the HCMV gB may be a variant gB, such as any of the variantHCMV gB glycoproteins disclosed herein, for example, any of the variantHCMV gB disclosed in the preceding paragraphs and in the Examples. Inany of these embodiments in which the vaccine comprises two or more HCMVgL proteins, the HCMV gL may be a variant gL, such as any of the variantHCMV gL glycoproteins disclosed herein, for example, any of the variantHCMV gL disclosed in the preceding paragraphs and in the Examples.

In certain embodiments in which the HCMV vaccine includes two or moreRNA polynucleotides having an open reading frame encoding two or moreHCMV antigenic polypeptides or an immunogenic fragment or epitopethereof (either encoded by a single RNA polynucleotide or encoded by twoor more RNA polynucleotides, for example, each protein encoded by aseparate RNA polynucleotide), the two or more HCMV proteins are avariant gB, for example, any of the variant gB polypeptides disclosedherein in the preceding paragraphs, and a HCMV protein selected from gH,gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides or immunogenicfragments or epitopes thereof. In some embodiments, the two or more HCMVproteins are a variant gH, for example, any of the variant gHpolypeptides disclosed herein in the preceding paragraphs, and a HCMVprotein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131Apolypeptides or immunogenic fragments or epitopes thereof. In someembodiments, the two or more HCMV proteins are a variant gH, forexample, any of the variant gH polypeptides disclosed herein in thepreceding paragraphs, and a HCMV protein selected from gH, gL, gO, gM,gN, UL128, UL130, and UL131A polypeptides or immunogenic fragments orepitopes thereof. In some embodiments in which the variant HCMV proteinsare variant HCMV gB, variant HCMV gL, and variant HCMV gH, the variantHCMV polypeptide is a truncated polypeptide selected from the followingtruncated polypeptides: lacks the hydrophobic membrane proximal domain;lacks the transmembrane domain; lacks the cytoplasmic domain; lacks twoor more of the hydrophobic membrane proximal, transmembrane, andcytoplasmic domains; and comprises only the ectodomain.

In some embodiments, the HCMV vaccine includes multimeric RNApolynucleotides having an open reading frame encoding at least one HCMVantigenic polypeptide or an immunogenic fragment or epitope thereof.Some embodiments of the present disclosure provide HCMV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment or epitope thereof, wherein the 5′UTR of the RNApolynucleotide comprises a patterned UTR. In some embodiments, thepatterned UTR has a repeating or alternating pattern, such as ABABAB orAABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, ormore than 3 times. In these patterns, each letter, A, B, or C representa different UTR at the nucleotide level. In some embodiments, the 5′UTRof the RNA polynucleotide (e.g., a first nucleic acid) has regions ofcomplementarity with a UTR of another RNA polynucleotide (a secondnucleic acid). For example, UTR nucleotide sequences of twopolynucleotides sought to be joined (e.g., in a multimeric molecule) canbe modified to include a region of complementarity such that the twoUTRs hybridize to form a multimeric molecule.

In some embodiments, the 5′UTR of an RNA polynucleotide encoding an HCMVantigenic polypeptide is modified to allow the formation of a multimericsequence. In some embodiments, the 5′UTR of an RNA polynucleotideencoding an HCMV protein selected from UL128, UL130, UL131A1 is modifiedto allow the formation of a multimeric sequence. In some embodiments,the 5′UTR of an RNA polynucleotide encoding an HCMV glycoprotein ismodified to allow the formation of a multimeric sequence. In someembodiments, the 5′UTR of an RNA polynucleotide encoding an HCMVglycoprotein selected from gH, gL, gB, gO, gM, and gN is modified toallow the formation of a multimeric sequence. In any of theseembodiments, the multimer may be a dimer, a trimer, pentamer, hexamer,heptamer, octamer nonamer, or decamer. Thus, in some embodiments, the5′UTR of an RNA polynucleotide encoding an HCMV protein selected fromgH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A1 is modified to allowthe formation of a dimer. In some embodiments, the 5′UTR of an RNApolynucleotide encoding an HCMV protein selected from gH, gL, gB, gO,gM, gN, UL128, UL130, and UL131A1 is modified to allow the formation ofa trimer. In some embodiments, the 5′UTR of an RNA polynucleotideencoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128,UL130, and UL131A1 is modified to allow the formation of a pentamer.Exemplary HCMV nucleic acids having modified 5′UTR sequence for theformation of a multimeric molecule (e.g., dimers, trimers, pentamers,etc) comprise SEQ ID Nos: 19-26.

In any of the above-described embodiments, the HCMV RNA polynucleotidesmay further comprise additional sequences, for example, one or morelinker sequences or one or more sequence tags, such as FLAG-tag andhistidine tag.

Some embodiments of the present disclosure provide HCMV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having asingle open reading frame encoding two or more (for example, two, three,four, five, or more) HCMV antigenic polypeptides or an immunogenicfragment or epitope thereof. Some embodiments of the present disclosureprovide HCMV vaccines that include at least one ribonucleic acid (RNA)polynucleotide having more than one open reading frame, for example,two, three, four, five or more open reading frames encoding two, three,four, five or more HCMV antigenic polypeptides. In either of theseembodiments, the at least one RNA polynucleotide may encode two or moreHCMV antigenic polypeptides selected from gH, gB, gL, gO, gM, gN, UL83,UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In someembodiments, the at least one RNA polynucleotide encodes UL83 and UL123.In some embodiments, the at least one RNA polynucleotide encodes gH andgL. In some embodiments, the at least one RNA polynucleotide encodesUL128, UL130, and UL131A. In some embodiments, the at least one RNApolynucleotide encodes gH, gL, UL128, UL130, and UL131A. In someembodiments, in which the at least one RNA polynucleotide has a singleopen reading frame encoding two or more (for example, two, three, four,five, or more) HCMV antigenic polypeptides, the RNA polynucleotidefurther comprises additional sequence, for example, a linker sequence ora sequence that aids in the processing of the HCMV RNA transcripts orpolypeptides, for example a cleavage site sequence. In some embodiments,the additional sequence may be a protease sequence, such as a furinsequence. In some embodiments, the additional sequence may beself-cleaving 2A peptide, such as a P2A, E2A, F2A, and T2A sequence. Insome embodiments, the linker sequences and cleavage site sequences areinterspersed between the sequences encoding HCMV polypeptides. In someembodiments, the RNA polynucleotide is encoded by SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence selected from any of SEQ ID NOs: 1-31,58, 60, 62, 64, 66, 68, and 108-113 and homologs having at least 80%(e.g., 85%, 90%, 95%, 98%, 99%) identity with a nucleic acid sequenceselected from SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113. Insome embodiments, at least one RNA polynucleotide is encoded by at leastone nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60,62, 64, 66, 68, and 108-113 and homologs having at least 90% (90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with anucleic acid sequence selected from SEQ ID NO:1-31, 58, 60, 62, 64, 66,68, and 108-113. In some embodiments, at least one RNA polynucleotide isencoded by at least one fragment of a nucleic acid sequence selectedfrom any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113 andhomologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identitywith a nucleic acid sequence selected from SEQ ID NO:1-31, 58, 60, 62,64, 66, 68, and 108-113. In some embodiments, at least one RNApolynucleotide is encoded by at least one nucleic acid sequence selectedfrom any of the nucleic acid sequences disclosed herein and homologshaving at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%) identitywith any of the nucleic acid sequences disclosed herein.

In any of the above-described embodiments in the preceding paragraphs,the HCMV RNA polynucleotides may further comprise additional sequences,for example, one or more linker sequences or one or more sequence tags,such as FLAG-tag and histidine tag.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide having at least 90% identity to the amino acidsequence of any of SEQ ID NOs: 32-52, 59, 61, 63, 65, 67, and 69. Insome embodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having at least 95% identity to the amino acid sequence ofany of SEQ ID Nos: 32-52, 59, 61, 63, 65, 67, and 69. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having at least 96% identity to the amino acid sequence ofany of SEQ ID Nos:32-52, 59, 61, 63, 65, 67, and 69. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having at least 97% identity to the amino acid sequence ofany of SEQ ID Nos: 32-52, 59, 61, 63, 65, 67, and 69. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having at least 98% identity to the amino acid sequence ofSEQ ID Nos: 32-52, 59, 61, 63, 65, 67, and 69. In some embodiments, atleast one RNA polynucleotide encodes an antigenic polypeptide having atleast 99% identity to the amino acid sequence of SEQ ID Nos: 32-52, 59,61, 63, 65, 67, and 69.

In some embodiments, the open reading from which the HCMV polypeptide isencoded is codon-optimized. In some embodiments, the at least one RNApolynucleotide encodes an antigenic protein of SEQ ID NO: 32, andwherein the RNA polynucleotide is codon optimized mRNA. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 33, and wherein the RNA polynucleotide is codonoptimized mRNA. In some embodiments, the at least one RNA polynucleotideencodes an antigenic protein of SEQ ID NO: 34, and wherein the RNApolynucleotide is codon optimized mRNA. In some embodiments, the atleast one RNA polynucleotide encodes an antigenic protein of SEQ ID NO:38, and wherein the RNA polynucleotide is codon optimized mRNA. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 40, and wherein the RNA polynucleotide is codonoptimized mRNA. In some embodiments, the at least one RNA polynucleotideencodes an antigenic protein of SEQ ID NO: 42, and wherein the RNApolynucleotide is codon optimized mRNA. In some embodiments, the atleast one RNA polynucleotide encodes an antigenic protein of SEQ ID NO:47, and wherein the RNA polynucleotide is codon optimized mRNA. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 50, and wherein the RNA polynucleotide is codonoptimized mRNA.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 32, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 32, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 33, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 33, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 34, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 34, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 38, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 38, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 40, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 40, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 42, and wherein the RNA polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO: 42, and wherein the RNA polynucleotide has greaterthan 80% identity to wild-type mRNA sequence, but does not includewild-type mRNA sequence.

In some embodiments, the at least one RNA polynucleotide is encoded by asequence selected from SEQ ID NO: 1-31 and includes at least onechemical modification.

In some embodiments, the HCMV vaccine is multivalent. In someembodiments, the RNA polynucleotide comprises a polynucleotide sequencederived from a virus strain or isolate selected from VR1814 VR6952,VR3480B1 (ganciclovir resistant), VR4760 (ganciclovir and foscarnetresistant), Towne, TB40/E, AD169, Merlin, and Toledo.

Some embodiments of the present disclosure provide a HCMV vaccine thatincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment thereof and at least one 5′ terminal cap. Insome embodiments, a 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.

Some embodiments of the present disclosure provide a HCMV vaccine thatincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment thereof, wherein the at least one ribonucleicacid (RNA) polynucleotide has at least one chemical modification. Insome embodiments, the at least one ribonucleic acid (RNA) polynucleotidefurther comprises a second chemical modification. In some embodiments,the at least one ribonucleic acid (RNA) polynucleotide having at leastone chemical modification has a 5′ terminal cap. In some embodiments,the at least one chemical modification is selected from pseudouridine,N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine and 2′-O-methyl uridine.

Some embodiments of the present disclosure provide a HCMV vaccine thatincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%,95%, 98%, 99%, 100%) of the uracil in the open reading frame have achemical modification, optionally wherein the vaccine is formulated in alipid nanoparticle. In some embodiments, 100% of the uracil in the openreading frame have a chemical modification. In some embodiments, achemical modification is in the 5-position of the uracil. In someembodiments, a chemical modification is a N1-methyl pseudouridine. Insome embodiments, a chemical modification is a N1-ethyl pseudouridine.

Some embodiments of the present disclosure provide a HCMVvaccine that isformulated within a cationic lipid nanoparticle. In some embodiments,the cationic lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid.

In some embodiments, a cationic lipid is an ionizable cationic lipid andthe non-cationic lipid is a neutral lipid, and the sterol is acholesterol. In some embodiments, a cationic lipid is selected from thegroup consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319),(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), andN,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

In some embodiments, the lipid is

In some embodiments, the lipid is

In some embodiments, the cationic lipid nanoparticle has a molar ratioof about 20-60% cationic lipid, about 5-25% non-cationic lipid, about25-55% sterol, and about 0.5-15% PEG-modified lipid. In someembodiments, the nanoparticle has a polydiversity value of less than0.4. In some embodiments, the nanoparticle has a net neutral charge at aneutral pH. In some embodiments, the nanoparticle has a mean diameter of50-200 nm.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, comprisingadministering to the subject a HCMV RNA vaccine in an amount effectiveto produce an antigen specific immune response. In some embodiments, anantigen specific immune response comprises a T cell response or a B cellresponse. In some embodiments, an antigen specific immune responsecomprises a T cell response and a B cell response. In some embodiments,a method of producing an antigen specific immune response involves asingle administration of the vaccine. In some embodiments, a methodfurther includes administering to the subject a booster dose of thevaccine. In some embodiments, a vaccine is administered to the subjectby intradermal or intramuscular injection.

Also provided herein are HCMV RNA vaccines for use in a method ofinducing an antigen specific immune response in a subject, the methodcomprising administering the vaccine to the subject in an amounteffective to produce an antigen specific immune response.

Further provided herein are uses of HCMV RNA vaccines in the manufactureof a medicament for use in a method of inducing an antigen specificimmune response in a subject, the method comprising administering thevaccine to the subject in an amount effective to produce an antigenspecific immune response.

Further provided herein are methods of preventing or treating HCMVinfection comprising administering to a subject the vaccine of thepresent disclosure.

The HCMV vaccine disclosed herein may be formulated in an effectiveamount to produce an antigen specific immune response in a subject.

In some embodiments, an anti-HCMV antigenic polypeptide antibody titerproduced in the subject is increased by at least 1 log relative to acontrol. In some embodiments, the anti-HCMV antigenic polypeptideantibody titer produced in the subject is increased by 1-3 log relativeto a control. In some embodiments, an anti-HCMV antigenic polypeptideantibody titer produced in the subject is increased at least 2 timesrelative to a control. In some embodiments, the anti-HCMV antigenicpolypeptide antibody titer produced in the subject is increased at least5 times relative to a control. In some embodiments, the anti-HCMVantigenic polypeptide antibody titer produced in the subject isincreased at least 10 times relative to a control. In some embodiments,the anti-HCMV antigenic polypeptide antibody titer produced in thesubject is increased 2-10 times relative to a control.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has not been administered HCMVvaccine.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered a liveattenuated or inactivated HCMV vaccine.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered arecombinant or purified HCMV protein vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 2-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 4-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 10-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 100-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 1000-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to a2-1000-fold reduction in the standard of care dose of a recombinant HCMVprotein vaccine, and wherein an anti-HCMV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-HCMV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified HCMV proteinvaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a total dose of 50-1000 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 100 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 400 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 500 μg administered to the subject atotal of two times.

Other aspects of the present disclosure provide methods of inducing anantigen specific immune response in a subject, including administeringto a subject the HCMV vaccine disclosed herein in an effective amount toproduce an antigen specific immune response in a subject.

In some embodiments, an anti-HCMV antigenic polypeptide antibody titerproduced in the subject is increased by at least 1 log relative to acontrol. In some embodiments, an anti-HCMV antigenic polypeptideantibody titer produced in the subject is increased by 1-3 log relativeto a control. In some embodiments, an anti-HCMV antigenic polypeptideantibody titer produced in the subject is increased at least 2 timesrelative to a control. In some embodiments, the anti-HCMV antigenicpolypeptide antibody titer produced in the subject is increased at least5 times relative to a control. In some embodiments, the anti-HCMVantigenic polypeptide antibody titer produced in the subject isincreased at least 10 times relative to a control. In some embodiments,the anti-HCMV antigenic polypeptide antibody titer produced in thesubject is increased 2-10 times relative to a control.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has not been administered HCMVvaccine.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered a liveattenuated or inactivated HCMV vaccine.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered arecombinant or purified HCMV protein vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 2-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant HCMV proteinvaccine or a live attenuated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 4-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 10-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 100-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 1000-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to a2-1000-fold reduction in the standard of care dose of a recombinant HCMVprotein vaccine, and wherein an anti-HCMV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-HCMV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified HCMV proteinvaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a total dose of 50-1000 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 100 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 400 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 500 μg administered to the subject atotal of two times.

Other aspects of the present disclosure provide HCMV vaccines containinga signal peptide linked to a HCMV antigenic polypeptide.

In some embodiments, the HCMV antigenic polypeptide is a HCMVglycoprotein or an antigenic fragment thereof. In some embodiments, theHCMV antigenic polypeptide is a HCMV gB, gM, gN, gH, gL, gO, UL 83,UL123, UL128, UL130, or UL131A protein or an antigenic fragment orepitope thereof. In some embodiments, the HCMV glycoprotein is selectedfrom HCMV gB, gM, gN, gH, gL, and gO.

In some embodiments, the HCMV glycoprotein is HCMV gH. In someembodiments, the HCMV glycoprotein is HCMV gL. In some embodiments, theHCMV glycoprotein is HCMV gB. In some embodiments, the HCMV protein isHCMV UL128. In some embodiments, the HCMV protein is HCMV UL130. In someembodiments, the HCMV protein is HCMV UL131A. In some embodiments, theHCMV protein is HCMV UL83. In some embodiments, the HCMV protein is HCMVUL123. In some embodiments, the HCMV glycoprotein is a variant HCMV gHpolypeptide. In some embodiments, the HCMV glycoprotein is a variantHCMV gL polypeptide. In some embodiments, the HCMV glycoprotein is avariant HCMV gB polypeptide.

In some embodiments, the signal peptide is an IgE signal peptide. Insome embodiments, the signal peptide is an IgE HC (Ig heavy chainepsilon-1) signal peptide. In some embodiments, the signal peptide hasthe amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 53).

In some embodiments, the signal peptide is an IgGκ signal peptide. Insome embodiments, the signal peptide has the amino acid sequenceMETPAQLLFLLLLWLPDTTG (SEQ ID NO: 54).

In some embodiments, the HCMV vaccine comprises at least one RNApolynucleotide encoding gH, gL, UL128, UL130, and UL131A, or antigenicfragments or epitopes thereof, and at least one RNA polynucleotideencoding gB, or an antigenic fragment or epitope thereof.

Further provided herein are uses of HCMV vaccines for prevention ofcongenital HCMV infection. Further provided herein are methods ofadministering HCMV vaccines to a women of child-bearing age.

Aspects of the invention relate to a human cytomegalovirus (HCMV)vaccine comprising: i) at least one RNA polynucleotide having one ormore open reading frames encoding HCMV antigenic polypeptides gH, gL,UL128, UL130, and/or UL131A, or antigenic fragments or epitopes thereof;ii) an RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide gB, or an antigenic fragment or epitope thereof;and iii) a pharmaceutically acceptable carrier or excipient.

In some embodiments, the HCMV vaccine comprises: an RNA polynucleotidehaving an open reading frame encoding HCMV antigenic polypeptide gH, oran antigenic fragment or epitope thereof; an RNA polynucleotide havingan open reading frame encoding HCMV antigenic polypeptide gL, or anantigenic fragment or epitope thereof; an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide UL128, or anantigenic fragment or epitope thereof; an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide UL130, or anantigenic fragment or epitope thereof; and an RNA polynucleotide havingan open reading frame encoding HCMV antigenic polypeptide UL131A, or anantigenic fragment or epitope thereof.

In some embodiments, at least one RNA polynucleotide has an open readingframe encoding two or more HCMV antigenic polypeptides, or antigenicfragments or epitopes thereof. In some embodiments, one or more of theopen reading frames is codon-optimized. In some embodiments, at leastone RNA polynucleotide is encoded by at least one nucleic acid sequenceselected from SEQ ID NOs: 58, 60, 62, 64, 66, 68, and 108-113. In someembodiments, at least one of the RNA polynucleotides encodes anantigenic polypeptide having at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identity to any of the aminoacid sequences of SEQ ID NOs: 59, 61, 63, 65, 67, and 69.

In some embodiments, at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO.: 59, wherein the RNA polynucleotide hasless than 80% identity to wild-type mRNA sequence or has greater than80% identity to wild-type mRNA sequence, but does not include wild-typemRNA sequence. In some embodiments, at least one RNA polynucleotideencodes an antigenic protein of SEQ ID NO.: 61, wherein the RNApolynucleotide has less than 80% identity to wild-type mRNA sequence orhas greater than 80% identity to wild-type mRNA sequence, but does notinclude wild-type mRNA sequence. In some embodiments, at least one RNApolynucleotide encodes an antigenic protein of SEQ ID NO.: 63, andwherein the RNA polynucleotide has less than 80% identity to wild-typemRNA sequence or has greater than 80% identity to wild-type mRNAsequence, but does not include wild-type mRNA sequence. In someembodiments, at least one RNA polynucleotide encodes an antigenicprotein of SEQ ID NO.: 65, and wherein the RNA polynucleotide has lessthan 80% identity to wild-type mRNA sequence or has greater than 80%identity to wild-type mRNA sequence, but does not include wild-type mRNAsequence. In some embodiments, at least one RNA polynucleotide encodesan antigenic protein of SEQ ID NO.: 67, and wherein the RNApolynucleotide has less than 80% identity to wild-type mRNA sequence orhas greater than 80% identity to wild-type mRNA sequence, but does notinclude wild-type mRNA sequence. In some embodiments, at least one RNApolynucleotide encodes an antigenic protein of SEQ ID NO.: 69, andwherein the RNA polynucleotide has less than 80% identity to wild-typemRNA sequenceor has greater than 80% identity to wild-type mRNAsequence, but does not include wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide includes at leastone chemical modification. In some embodiments, the vaccine ismultivalent. In some embodiments, the RNA polynucleotide comprises apolynucleotide sequence derived from a virus strain or isolate selectedfrom VR1814, VR6952, VR3480B1, VR4760, Towne, TB40/E, AD169, Merlin, andToledo.

In some embodiments, the HCMV vaccine further comprises a secondchemical modification. In some embodiments, the chemical modification isselected from the group consisting of pseudouridine,N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, and 2′-O-methyl uridine.

In some embodiments, 80% of the uracil in the open reading frame have achemical modification. In some embodiments, 100% of the uracil in theopen reading frame have a chemical modification. In some embodiments,the chemical modification is in the 5-position of the uracil. In someembodiments, the chemical modification is N1-methylpseudouridine. Insome embodiments, the chemical modification is N1-ethylpseudouridine.

In some embodiments, the vaccine is formulated within a cationic lipidnanoparticle. In some embodiments, the cationic lipid nanoparticlecomprises a cationic lipid, a PEG-modified lipid, a sterol and anon-cationic lipid. In some embodiments, the cationic lipid is anionizable cationic lipid and the non-cationic lipid is a neutral lipid,and the sterol is a cholesterol. In some embodiments, the cationic lipidis selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In someembodiments, the cationic lipid nanoparticle has a molar ratio of about20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55%sterol, and about 0.5-15% PEG-modified lipid.

In some embodiments, the nanoparticle has a polydiversity value of lessthan 0.4. In some embodiments, the nanoparticle has a net neutral chargeat a neutral pH. In some embodiments, the nanoparticle has a meandiameter of 50-200 nm. Aspects of the invention relate to methods ofinducing an antigen specific immune response in a subject, comprisingadministering any of the vaccines described herein to the subject in aneffective amount to produce an antigen specific immune response. In someembodiments, the antigen specific immune response comprises a T cellresponse. In some embodiments, the antigen specific immune responsecomprises a B cell response. In some embodiments, the antigen specificimmune response comprises a T cell response and a B cell response. Insome embodiments, the method of producing an antigen specific immuneresponse involves a single administration of the vaccine. In someembodiments, methods further comprise administering a booster dose ofthe vaccine. In some embodiments, the vaccine is administered to thesubject by intradermal or intramuscular injection.

Aspects of the invention relate to HCMV vaccines as described herein foruse in a method of inducing an antigen specific immune response in asubject, the method comprising administering the vaccine to the subjectin an effective amount to produce an antigen specific immune response.

Aspects of the invention relate to the use of an HCMV vaccine describedherein in the manufacture of a medicament for use in a method ofinducing an antigen specific immune response in a subject, the methodcomprising administering the vaccine to the subject in an effectiveamount to produce an antigen specific immune response.

Aspects of the invention relate to methods of preventing or treatingHCMV infection comprising administering to a subject a vaccine describedherein.

Aspects of the invention relate to HCMV vaccines described hereinformulated in an effective amount to produce an antigen specific immuneresponse in a subject. In some embodiments, an anti-HCMV antigenicpolypeptide antibody titer produced in the subject is increased by atleast 1 log relative to a control, or by 1-3 log relative to a control.In some embodiments, an anti-HCMV antigenic polypeptide antibody titerproduced in the subject is increased at least 2 times relative to acontrol, at least 5 times relative to a control, at least 10 timesrelative to a control, or 2-10 times relative to a control.

In some embodiments, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has not been administered HCMVvaccine. In some embodiments, the control is an anti-HCMV antigenicpolypeptide antibody titer produced in a subject who has beenadministered a live attenuated or inactivated HCMV vaccine. In someembodiments, the control is an anti-HCMV antigenic polypeptide antibodytiter produced in a subject who has been administered a recombinant orpurified HCMV protein vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 2-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 4-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 10-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 100-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 1000-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine, and wherein an anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a dose equivalent to a2-1000-fold reduction in the standard of care dose of a recombinant HCMVprotein vaccine, and wherein an anti-HCMV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-HCMV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified HCMV proteinvaccine or a live attenuated or inactivated HCMV vaccine.

In some embodiments, the effective amount is a total dose of 50-1000 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 100 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 400 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 500 μg administered to the subject atotal of two times.

In some embodiments of methods disclosed herein, an anti-HCMV antigenicpolypeptide antibody is produced in the subject and wherein the titer ofthe anti-HCMV antigenic polypeptide antibody is increased by at least 1log relative to a control. In some embodiments, the anti-HCMV antigenicpolypeptide antibody titer produced in the subject is increased by 1-3log relative to a control.

In some embodiments of methods disclosed herein, the anti-HCMV antigenicpolypeptide antibody titer produced in the subject is increased at least2 times relative to a control, at least 5 times relative to a control,at least 10 times relative to a control, or 2-10 times relative to acontrol.

In some embodiments of methods disclosed herein, the control is ananti-HCMV antigenic polypeptide antibody titer produced in a subject whohas not been administered HCMV vaccine. In some embodiments of methodsdisclosed herein, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered a liveattenuated or inactivated HCMV vaccine. In some embodiments of methodsdisclosed herein, the control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject who has been administered arecombinant or purified HCMV protein vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to an at least 2-fold reduction in the standard ofcare dose of a recombinant HCMV protein vaccine, and wherein ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant HCMV protein vaccine or a live attenuated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to an at least 4-fold reduction in the standard ofcare dose of a recombinant HCMV protein vaccine, and wherein ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to an at least 10-fold reduction in the standard ofcare dose of a recombinant HCMV protein vaccine, and wherein ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to an at least 100-fold reduction in the standard ofcare dose of a recombinant HCMV protein vaccine, and wherein ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to an at least 1000-fold reduction in the standard ofcare dose of a recombinant HCMV protein vaccine, and wherein ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa dose equivalent to a 2-1000-fold reduction in the standard of caredose of a recombinant HCMV protein vaccine, and wherein an anti-HCMVantigenic polypeptide antibody titer produced in the subject isequivalent to an anti-HCMV antigenic polypeptide antibody titer producedin a control subject administered the standard of care dose of arecombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments of methods disclosed herein, the effective amount isa total dose of 50-1000 μg. In some embodiments of methods disclosedherein, the effective amount is a total dose of 100 μg. In someembodiments of methods disclosed herein, the effective amount is a doseof 25 μg administered to the subject a total of two times. In someembodiments of methods disclosed herein, the effective amount is a doseof 100 μg administered to the subject a total of two times. In someembodiments of methods disclosed herein, the effective amount is a doseof 400 μg administered to the subject a total of two times. In someembodiments of methods disclosed herein, the effective amount is a doseof 500 μg administered to the subject a total of two times.

Aspects of the invention relate to an HCMV vaccine, comprising: i) HCMVantigenic polypeptides gH, gL, UL128, UL130, and/or UL131A, or antigenicfragments or epitopes thereof; and ii) HCMV antigenic polypeptide gB, oran antigenic fragment or epitope thereof; wherein one or more of theHCMV antigenic polypeptides comprises a signal sequence linked to theHCMV antigenic polypeptide.

In some embodiments, the signal peptide is an IgE signal peptide. Insome embodiments, the signal peptide is an IgE HC (Ig heavy chainepsilon-1) signal peptide. In some embodiments, the signal peptide hasthe amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 53). In someembodiments, the signal peptide is an IgGκ signal peptide. In someembodiments, the signal peptide has the amino acid sequenceMETPAQLLFLLLLWLPDTTG (SEQ ID NO: 54).

In some embodiments, the subject is a woman of child-bearing age.

Aspects of the invention relate to methods of preventing congenital HCMVinfection comprising administering to a woman of child-bearing age atherapeutically effective amount of a human cytomegalovirus (HCMV)vaccine comprising: i) at least one RNA polynucleotide having one ormore open reading frames encoding HCMV antigenic polypeptides gH, gL,UL128, UL130, and/or UL131A, or antigenic fragments or epitopes thereof,ii) an RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide gB, or an antigenic fragment or epitope thereof,and iv) a pharmaceutically acceptable carrier or excipient.

In some embodiments of methods disclosed herein, the vaccine comprises:an RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide gH, or an antigenic fragment or epitope thereof;an RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide gL, or an antigenic fragment or epitope thereof,an RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide UL128, or an antigenic fragment or epitopethereof; an RNA polynucleotide having an open reading frame encodingHCMV antigenic polypeptide UL130, or an antigenic fragment or epitopethereof; and an RNA polynucleotide having an open reading frame encodingUL131, or an antigenic fragment or epitope thereof.

In some embodiments the nucleic acid vaccines described herein arechemically modified. In other embodiments the nucleic acid vaccines areunmodified.

Yet other aspects provide compositions for and methods of vaccinating asubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a first antigenic polypeptide, wherein the RNA polynucleotidedoes not include a stabilization element, and wherein an adjuvant is notcoformulated or co-administered with the vaccine.

In other aspects the invention is a composition for or method ofvaccinating a subject comprising administering to the subject a nucleicacid vaccine comprising one or more RNA polynucleotides having an openreading frame encoding a first antigenic polypeptide wherein a dosage ofbetween 10 μg/kg and 400 μg/kg of the nucleic acid vaccine isadministered to the subject. In some embodiments the dosage of the RNApolynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg,100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg,80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg,300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or300-400 μg per dose. In some embodiments, the nucleic acid vaccine isadministered to the subject by intradermal or intramuscular injection.In some embodiments, the nucleic acid vaccine is administered to thesubject on day zero. In some embodiments, a second dose of the nucleicacid vaccine is administered to the subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 100 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 50 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 75 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 150 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 400 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 200 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, the RNA polynucleotide accumulates at a 100 foldhigher level in the local lymph node in comparison with the distal lymphnode. In other embodiments the nucleic acid vaccine is chemicallymodified and in other embodiments the nucleic acid vaccine is notchemically modified.

Aspects of the invention provide a nucleic acid vaccine comprising oneor more RNA polynucleotides having an open reading frame encoding afirst antigenic polypeptide, wherein the RNA polynucleotide does notinclude a stabilization element, and a pharmaceutically acceptablecarrier or excipient, wherein an adjuvant is not included in thevaccine. In some embodiments, the stabilization element is a histonestem-loop. In some embodiments, the stabilization element is a nucleicacid sequence having increased GC content relative to wild typesequence.

Aspects of the invention provide nucleic acid vaccines comprising one ormore RNA polynucleotides having an open reading frame encoding a firstantigenic polypeptide, wherein the RNA polynucleotide is present in theformulation for in vivo administration to a host, which confers anantibody titer superior to the criterion for seroprotection for thefirst antigen for an acceptable percentage of human subjects. In someembodiments, the antibody titer produced by the mRNA vaccines of theinvention is a neutralizing antibody titer. In some embodiments theneutralizing antibody titer is greater than a protein vaccine. In otherembodiments the neutralizing antibody titer produced by the mRNAvaccines of the invention is greater than an adjuvanted protein vaccine.In yet other embodiments the neutralizing antibody titer produced by themRNA vaccines of the invention is 1,000-10,000, 1,200-10,000,1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000,2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000,3,000-4,000, or 2,000-2,500. A neutralization titer is typicallyexpressed as the highest serum dilution required to achieve a 50%reduction in the number of plaques.

Also provided are nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide, wherein the RNA polynucleotide is present in a formulationfor in vivo administration to a host for eliciting a longer lasting highantibody titer than an antibody titer elicited by an mRNA vaccine havinga stabilizing element or formulated with an adjuvant and encoding thefirst antigenic polypeptide. In some embodiments, the RNA polynucleotideis formulated to produce a neutralizing antibodies within one week of asingle administration. In some embodiments, the adjuvant is selectedfrom a cationic peptide and an immunostimulatory nucleic acid. In someembodiments, the cationic peptide is protamine.

Aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no nucleotide modification, the openreading frame encoding a first antigenic polypeptide, wherein the RNApolynucleotide is present in the formulation for in vivo administrationto a host such that the level of antigen expression in the hostsignificantly exceeds a level of antigen expression produced by an mRNAvaccine having a stabilizing element or formulated with an adjuvant andencoding the first antigenic polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no nucleotide modification, the openreading frame encoding a first antigenic polypeptide, wherein thevaccine has at least 10 fold less RNA polynucleotide than is requiredfor an unmodified mRNA vaccine to produce an equivalent antibody titer.In some embodiments, the RNA polynucleotide is present in a dosage of25-100 micrograms. Aspects of the invention also provide a unit of usevaccine, comprising between bug and 400 ug of one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no nucleotide modification, the openreading frame encoding a first antigenic polypeptide, and apharmaceutically acceptable carrier or excipient, formulated fordelivery to a human subject. In some embodiments, the vaccine furthercomprises a cationic lipid nanoparticle.

Aspects of the invention provide methods of creating, maintaining orrestoring antigenic memory to a virus strain in an individual orpopulation of individuals comprising administering to said individual orpopulation an antigenic memory booster nucleic acid vaccine comprising(a) at least one RNA polynucleotide, said polynucleotide comprising atleast one chemical modification or optionally no nucleotide modificationand two or more codon-optimized open reading frames, said open readingframes encoding a set of reference antigenic polypeptides, and (b)optionally a pharmaceutically acceptable carrier or excipient. In someembodiments, the vaccine is administered to the individual via a routeselected from the group consisting of intramuscular administration,intradermal administration and subcutaneous administration. In someembodiments, the administering step comprises contacting a muscle tissueof the subject with a device suitable for injection of the composition.In some embodiments, the administering step comprises contacting amuscle tissue of the subject with a device suitable for injection of thecomposition in combination with electroporation.

Aspects of the invention provide methods of vaccinating a subjectcomprising administering to the subject a single dosage of between 25ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide in an effective amount to vaccinate the subject. Otheraspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification, the open reading frame encoding a first antigenicpolypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine toproduce an equivalent antibody titer. In some embodiments, the RNApolynucleotide is present in a dosage of 25-100 micrograms.

Other aspects provide nucleic acid vaccines comprising an LNP formulatedRNA polynucleotide having an open reading frame comprising no nucleotidemodifications (unmodified), the open reading frame encoding a firstantigenic polypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine notformulated in a LNP to produce an equivalent antibody titer. In someembodiments, the RNA polynucleotide is present in a dosage of 25-100micrograms.

The data presented in the Examples demonstrate significant enhancedimmune responses using the formulations of the invention. Surprisingly,in contrast to prior art reports that it was preferable to usechemically unmodified mRNA formulated in a carrier for the production ofvaccines, it is described herein that chemically modified mRNA-LNPvaccines require a much lower effective mRNA dose than unmodified mRNA,i.e., tenfold less than unmodified mRNA when formulated in carriersother than LNP. Both the chemically modified and unmodified RNA vaccinesof the invention produce better immune responses than mRNA vaccinesformulated in a different lipid carrier.

In other aspects the invention encompasses a method of treating anelderly subject age 60 years or older comprising administering to thesubject a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding an antigenicpolypeptide in an effective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating a youngsubject age 17 years or younger comprising administering to the subjecta nucleic acid vaccine comprising one or more RNA polynucleotides havingan open reading frame encoding an antigenic polypeptide in an effectiveamount to vaccinate the subject.

In other aspects the invention encompasses a method of treating an adultsubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding an antigenic polypeptide in an effective amount to vaccinatethe subject.

In some aspects the invention is a method of vaccinating a subject witha combination vaccine including at least two nucleic acid sequencesencoding antigens wherein the dosage for the vaccine is a combinedtherapeutic dosage wherein the dosage of each individual nucleic acidencoding an antigen is a sub therapeutic dosage. In some embodiments,the combined dosage is 25 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 100 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodimentsthe combined dosage is 50 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 75 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 150 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 400 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the sub therapeutic dosage of each individual nucleic acid encoding anantigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 micrograms. In other embodiments the nucleic acid vaccineis chemically modified and in other embodiments the nucleic acid vaccineis not chemically modified.

In some embodiments, the RNA polynucleotide is one of SEQ ID NO: 1-6,58, 60, 62, 64, 66. 68, and 108-113 and includes at least one chemicalmodification. In other embodiments the RNA polynucleotide is one of SEQID NO: 1-6, 58, 60, 62, 64, 66, 68, and 108-113 and does not include anynucleotide modifications, or is unmodified. In yet other embodiments theat least one RNA polynucleotide encodes an antigenic protein of any ofSEQ ID NO: 7-12, 59, 61, 63, 65, 67, and 69 and includes at least onechemical modification. In other embodiments the RNA polynucleotideencodes an antigenic protein of any of SEQ ID NO: 7-12, 59, 61, 63, 65,67, and 69 and does not include any nucleotide modifications, or isunmodified.

In preferred aspects, vaccines of the invention (e.g., LNP-encapsulatedmRNA vaccines) produce prophylactically- and/ortherapeutically-efficacious levels, concentrations and/or titers ofantigen-specific antibodies in the blood or serum of a vaccinatedsubject. As defined herein, the term antibody titer refers to the amountof antigen-specific antibody produces in s subject, e.g., a humansubject. In exemplary embodiments, antibody titer is expressed as theinverse of the greatest dilution (in a serial dilution) that still givesa positive result. In exemplary embodiments, antibody titer isdetermined or measured by enzyme-linked immunosorbent assay (ELISA). Inexemplary embodiments, antibody titer is determined or measured byneutralization assay, e.g., by microneutralization assay. In certainaspects, antibody titer measurement is expressed as a ratio, such as1:40, 1:100, etc.

In exemplary embodiments of the invention, an efficacious vaccineproduces an antibody titer of greater than 1:40, greater that 1:100,greater than 1:400, greater than 1:1000, greater than 1:2000, greaterthan 1:3000, greater than 1:4000, greater than 1:500, greater than1:6000, greater than 1:7500, greater than 1:10000. In exemplaryembodiments, the antibody titer is produced or reached by 10 daysfollowing vaccination, by 20 days following vaccination, by 30 daysfollowing vaccination, by 40 days following vaccination, or by 50 ormore days following vaccination. In exemplary embodiments, the titer isproduced or reached following a single dose of vaccine administered tothe subject. In other embodiments, the titer is produced or reachedfollowing multiple doses, e.g., following a first and a second dose(e.g., a booster dose.)

In exemplary aspects of the invention, antigen-specific antibodies aremeasured in units of μg/ml or are measured in units of IU/L(International Units per liter) or mIU/ml (milli International Units perml). In exemplary embodiments of the invention, an efficacious vaccineproduces >0.5 μg/ml, >0.1 μg/ml, >0.2 μg/ml, >0.35 μg/ml, >0.5 μg/ml, >1μg/ml, >2 μg/ml, >5 μg/ml or >10 μg/ml. In exemplary embodiments of theinvention, an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml. Inexemplary embodiments, the antibody level or concentration is producedor reached by 10 days following vaccination, by 20 days followingvaccination, by 30 days following vaccination, by 40 days followingvaccination, or by 50 or more days following vaccination. In exemplaryembodiments, the level or concentration is produced or reached followinga single dose of vaccine administered to the subject. In otherembodiments, the level or concentration is produced or reached followingmultiple doses, e.g., following a first and a second dose (e.g., abooster dose.) In exemplary embodiments, antibody level or concentrationis determined or measured by enzyme-linked immunosorbent assay (ELISA).In exemplary embodiments, antibody level or concentration is determinedor measured by neutralization assay, e.g., by microneutralization assay.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIGS. 1A-1C depict different protein complexes formed by hCMV proteins.The tropism of hCMV is dictated by distinct protein complexes. FIG. 1Ashows the gH/gL/gB complex that mediates the entry of hCMV intofibroblasts. FIG. 1B shows the pentameric complex containinggH/gL/UL128/UL130/UL131A. Such a pentameric complex mediates the entryof hCMV into epithelial cells, endothelial cells, monocytes, anddendritic cells. FIG. 1C, which is adapted from Macagno et al. (2010) J.Virology 84(2):1005-13 shows the hCMV pentameric complex(gH/gL/UL128/UL130/UL131A) further in complex with antibodies specificfor the protein components of the pentameric complex: 8121(anti-pentamer), 3G16 (anti-gH), 15D8 (anti-UL128), 7113(anti-UL128/UL130/UL131A), and 10P3 (anti-gL).

FIGS. 2A-2D show that delivery of pre-mixed mRNAs encoding the varioussubunits of hCMV pentamer leads to surface expression of the pentamericcomplex in HeLa cells. FIG. 2A shows the surface expression of gH. FIG.2B shows the surface expression of UL128/UL130/UL131A. FIG. 2C shows thesurface expression of UL128. FIG. 2D shows the surface expression of thepentamer.

FIGS. 3A-3B show that the hCMV pentameric complex does not express onthe cell surface in the absence of one of the core subunits. mRNAsencoding all or some of the subunits in the pentamer were expressed inHeLa cells and the surface expression of the pentamer was detected by ananti-pentamer antibody (8I21). Surface expression of the pentamer wasonly detected at high levels when all the core subunits were expressed.

FIGS. 4A-B shows the dimerization of gH-gL is sufficient to lead tosurface expression of gH. The anti-gH antibody (3G16) was used for thedetection of gH on the cell surface. When gH and gL were co-expressed, asimilar level of gH was detected on the surface of HeLa cells as whenall subunits in the pentameric complex were expressed. When gH wasexpressed alone, very little gH was detected on the surface of thetransfected HeLa cells.

FIGS. 5A-5D show the intracellular and surface expression of hCMVantigen gB. The mRNA encoding gB was expressed both intracellularly andon the cell surface (FIGS. 5A-5C). Both gB precursor and theproteolytically processed, mature gB, were detected by anti-gBantibodies in an immunoblot (FIG. 5D). “*” indicates that the lane wasoverloaded.

FIG. 6 shows an immunogenicity study of the hCMV pentameric complex mRNAvaccine constructs. Mice were vaccinated according to the vaccinationschedule with indicated dosages of the mRNAs. High titers ofanti-pentamer antibodies were detected in mice serum following theimmunization. Different formulations of the pentamer mRNAs producedcomparable levels of antibodies. A third immunization did not lead toboosting of antibody production.

FIG. 7 shows an immunogenicity study of the hCMV gB mRNA vaccineconstruct, with or without the pentameric complex mRNA constructs. gBmRNA constructs produced similar IgG titers as the gB protein/MF59antigens after 3 immunizations. A boost in IgG production was observedafter the third immunization. Addition of pentameric mRNA constructs didnot interefere with the induction of anti-gB IgG.

FIG. 8 shows a neutralization study of the hCMV pentameric complex mRNAvaccine constructs in the epithelial cell line ARPE-19. IE1 staining ininfected ARPE-19 cells is demonstrated. Immunization with hCMVpentameric complex mRNA vaccine constructs elicits highly potentneutralizing antibodies in mice. Neutralizing antibody titer (1:25600)in mice serum at day 41 (3 weeks post second immunization) was able toneutrzalize the hCMV clinical isolate VR1814 in ARPE-19 cells.

FIG. 9 shows a measurement of hCMV neutralization IgG tiers in ARPE-19cells infected with the hCMV clinical isolate strain VR1814. See alsoTable 5.

FIGS. 10A-10B show the surface expression in HeLa cells of the hCMVpentameric complex (gH-gL-UL128-UL130-UL131A) encoded by thefirst-generation pentameric constructs described herein (referred to as“old”) and second-generation pentameric constructs also described herein(referred to as “new”). The sequences of the mRNAs within the secondgeneration constructs are provided in Table 6, corresponding to SEQ IDNOs: 58-69. FIGS. 10A and 10C shows the results of afluorescence-activated cell (FACS) sorting experiment detecting thesurface expression of the pentameric complex using the 8121(anti-pentamer) antibodies. Surface expression of the pentameric complexis indicated by the emerging fluorescent cell population. FIGS. 10B and10D shows the quantification of the FACS experiment.

FIGS. 11A-11E depicts Western blots showing the expression of thesubunits of the hCMV pentameric complex (gH, gL, UL128, UL130, andUL131A) encoded by the first generation pentameric constructs describedherein (referred to as “old”) and second-generation pentamericconstructs also described herein (referred to as “new”).

FIG. 12 shows that immunization with the pentameric mRNA complex elicitshigh titers of antibodies that are maintained up to several months. Animmunogenicity study of the hCMV pentameric complex mRNA vaccineconstructs is shown. Balb/c mice were vaccinated according to thevaccination schedule with indicated dosages of the mRNAs (lower panel).Mice serum IgG titers were measured at days 20, 41, 62, 92, and 123 postimmunization. hCMV pentamer coated plates were used to measure the serumIgG titer. High titers of anti-pentamer antibodies were detected in theserum of the immunized mice.

FIG. 13 shows that hCMV mRNA vaccine constructs elicited higherneutralizing antibody titers in mice than CytoGam®, a hyperimmune serumused clinically for prophylaxis of hCMV. Balb/c mice were vaccinatedaccording to the vaccination schedule with indicated dosages of themRNAs (lower panel). Neutralizing antibody titers in mice serum weremeasured at days 42, 122, 152, and 182 post immunization, with ARPE-19epithelial cells infected with the hCMV clinical isolate VR1814. Hightiters of neutralizing antibodies induced by the hCMV pentameric complexmRNA vaccine were maintained up to 6 months.

FIG. 14 is a graph showing the neutralizing antibody titers induced inmice by hCMV pentameric complex mRNA vaccine constructs. Balb/c micewere vaccinated according to the vaccination schedule with indicateddosages of the mRNAs (lower panel). Neutralizing antibody titers in miceserum were measured at days 42, 62, and 182 post immunization, withHEL299 fibroblast cells infected with 500-2000 pfu of hCMV AD169 strain.

FIG. 15 is a schematic representation of pentametic subunits linked by aself-cleaving 2A peptide (e.g., as described in Kim et al., PLoS ONE6(4): e18556, 2011).

FIG. 16 is a Western blot showing that gH and gL linked by the 2Apeptide underwent efficient self-cleavage to generate individual gH andgL subunits.

FIG. 17 is a graph showing that the individual gH and gL subunitsgenerated from self-cleavage of the 2A peptide linked were able todimerize and translocate to the cell surface.

FIGS. 18A-B demonstrates high and sustained titers of anti-pentamerbinding and neutralizing antibodies in mice. FIG. 18A depicts a graphshowing anti-pentamer antibody titers. Equimolar and equal massformulations of the pentameric mRNAs were compared and were found to beequally effective. FIG. 18B depicts a graph showing neutralizing titersmeasured on ARPE19 epithelial cells infected with hCMV strain VR1814.Equimolar and equal mass formulations of the pentameric mRNAs werecompared and were found to be equally effective. Neutralizing titerswere found to be approximately 25 fold higher than CytoGam®.

FIGS. 19A-C demonstrates that neutralization activity against epithelialcell infection is dependent on anti-pentamer antibodies. FIG. 19A showsthat the depleting protein was either the pentamer or a gH/gL dimer.FIG. 19B and FIG. 19C depict graphs showing neutralization. FIG. 19Bshows neutralization by sera from mice immunized with the pentamer orwith a gH/gL dimer. FIG. 19C shows neutralization by CytoGam® combinedwith the pentamer or with a gH/gL.

FIGS. 20A-20B are graphs showing the immunogenicity of second generationhCMV mRNA vaccine constructs formulated with Compound 25 lipids. Thesecond generation mRNA constructs encoding the pentamer and gB inducedpentamer-specific antibodies (FIG. 20A) and gB-specific antibodies (FIG.20B) as early as 20 days post first immunization. The pentamer-specificand gB-specific antibody titers continue to increase in mice after theboost dose.

FIG. 21 is a graph showing that a 3 μg total dose of HCMV mRNA vaccineconstructs encoding the pentameric complex elicited higherneutralization antibody titers than CytoGam®, a hyperimmune serum usedclinically for prophylaxis of hCMV.

FIG. 22 is a schematic showing an exemplary linking region, wherein X isany nucleic acid sequence of 0-100 nucleotides and A and B arecomplementary parts, which are complementary to one or more othernucleic acids.

FIG. 23 shows an example of a stabilizing nucleic acid with thefollowing structure: L₁X₁L₂X₂L₃X₃L₄X₄L₅X₅L₆X₆L₇, wherein L is a nucleicacid sequence complementary to a linking region and wherein x is anynucleic acid sequence 0-50 nucleotides in length.

FIG. 24 shows the immunization and bleed schedule corresponding to Table4.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include polynucleotide encoding a human cytomegalovirus (HCMV)antigen. The human cytomegalovirus (HCMV) is a ubiquitousdouble-stranded DNA virus belonging to the Herpes virus family. HCMV ismade up of a DNA core, an outer capsid and covered by a lipid membrane(envelope) which incorporates virus specific glycoproteins. The diameteris around 150-200 nm. Genomes are linear and non-segmented, around 200kb in length. Viral replication is nuclear, and is lysogenic.Replication is dsDNA bidirectional replication.

HCMV can infect a wide range of mammalian cells, which correlates withits ability to infect most organs and tissues. Entry into the host cellis achieved by attachment of the viral glycoproteins to host cellreceptors, which mediates endocytosis. HCMV displays a broad host cellrange, with the ability to infect several cell types, such asendothelial cells, epithelial cells, smooth muscle cells, fibroblasts,leukocytes, and dendritic cells. This broad cellular tropism suggeststhat HCMV may bind a number of receptors or a common surface molecule.

HCMV envelopment is very complicated and comprises more than 20glycoproteins which may be the reason for broad cellular tropism ofHCMV. HCMV particles contain at least four major glycoprotein complexes,all of which are involved in HCMV infection, which requires initialinteraction with the cell surface through binding to heparin sulfateproteoglycans and possibly other surface receptors.

The gCI complex is comprised of dimeric molecules of the glycoproteingB. Each 160-kDa monomer is cleaved to generate a 116-kDa surface unitlinked by disulfide bonds to a 55-kDa transmembrane component. Someantibodies immunospecific for gB inhibit the attachment of virions tocells, whereas others block the fusion of infected cells, suggestingthat the gB protein might execute multiple functions at the start ofinfection. Studies have confirmed that glycoprotein B (gB) facilitatesHCMV entry into cells by binding receptors and mediating membranefusion. Several cellular membrane proteins interact with gB, whichinteractions likely facilitate entry and activate cellular signalingpathways.

The gCII complex is the most abundant of the glycoprotein complexes andis a heterodimer consisting of glycoproteins gM and gN. The complexbinds to heparan sulfate proteoglycans, suggesting it might contributeto the initial interaction of the virion with the cell surface. It mayalso perform a structural role during virion assembly/envelopment,similar to the gM-gN complex found in some α-herpesviruses.

The gCIII complex is a trimer comprised of glycoproteins gH, gL, gOwhich are covalently linked by disulfide bonds. All known herpesvirusesencode gH-gL heterodimers, which mediate fusion of the virion envelopewith the cell membrane. Antibodies specific for human CMV gH do notaffect virus attachment but block penetration and cell-to-celltransmission. A gO-deficient mutant of HCMV (strain AD169) shows asignificant growth defect.

HCMV proteins UL128, UL130, and UL131A assemble with gH and gL proteinsto form a heterologous pentameric complex, designated gH/gL/UL128-131A,found on the surface of the HCMV. Natural variants and deletion andmutational analyses have implicated proteins of the gH/gL/UL128-131Acomplex with the ability to infect certain cell types, including forexample, endothelial cells, epithelial cells, and leukocytes.

HCMV enters cells by fusing its envelope with either the plasma membrane(fibroblasts) or the endosomal membrane (epithelial and endothelialcells). HCMV initiates cell entry by attaching to the cell surfaceheparan sulfate proteoglycans using envelope glycoprotein M (gM) or gB.This step is followed by interaction with cell surface receptors thattrigger entry or initiate intracellular signaling. The entry receptorfunction is provided by gH/gL glycoprotein complexes. Different gH/gLcomplexes are known to facilitate entry into epithelial cells,endothelial cells, or fibroblasts. For example, while entry intofibroblasts requires gH/gL heterodimer, entry into epithelial andendothelial cells requires the pentameric complexgH/gL/UL128/UL130/UL131 in addition to gH/gL. Thus, different gH/gLcomplexes engage distinct entry receptors on epithelial/endothelialcells and fibroblasts. Receptor engagement is followed by membranefusion, a process mediated by gB and gH/gL. Early antibody studies havesupported critical roles for both gB and gH/gL in HCMV entry. gB isessential for entry and cell spread. gB and gH/gL are necessary andsufficient for cell fusion and thus constitute the “core fusionmachinery” of HCMV, which is conserved among other herpesviruses.

Thus, the four glycoprotein complexes play a crucial role in viralattachment, binding, fusion and entry into the host cell.

Studies involving the gH/gL/UL128-131A complex have shown that HCMVglycoproteins gB, gH, gL, gM, and gN, as well as UL128, UL130, andUL131A proteins, are antigenic and involved in the immunostimulatoryresponse in a variety of cell types. Moreover, UL128, UL130, and UL131Agenes are relatively conserved among HCMV isolates and thereforerepresent an attractive target for vaccination. Furthermore, recentstudies have shown that antibodies to epitopes within the pentamericgH/gL/UL128-131 complex neutralize entry into endothelial, epithelial,and other cell types, thus blocking the ability of HCMV to infectseveral cell types.

HCMV envelope glycoprotein complexes (gCI, II, III, gH/gL/UL128-131A)represent major antigenic targets of antiviral immune responses.Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include polynucleotide encoding a HCMV antigen, in particular anHCMV antigen from one of the HCMV glycoprotein complexes. Embodiments ofthe present disclosure provide RNA (e.g., mRNA) vaccines that include atleast one polynucleotide encoding at least one HCMV antigenicpolypeptide. The HCMV RNA vaccines provided herein may be used to inducea balanced immune response, comprising both cellular and humoralimmunity, without many of the risks associated with DNA vaccines andlive attenuated vaccines.

The entire contents of International Application No. PCT/US2015/027400(WO 2015/164674), entitled “Nucleic Acid Vaccines,” is incorporatedherein by reference. It has been discovered that the mRNA vaccinesdescribed herein are superior to current vaccines in several ways.First, the lipid nanoparticle (LNP) delivery is superior to otherformulations including a protamine base approach described in theliterature and no additional adjuvants are to be necessary. The use ofLNPs enables the effective delivery of chemically modified or unmodifiedmRNA vaccines. Additionally it has been demonstrated herein that bothmodified and unmodified LNP formulated mRNA vaccines were superior toconventional vaccines by a significant degree. In some embodiments themRNA vaccines of the invention are superior to conventional vaccines bya factor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500fold or 1,000 fold.

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines have not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the invention a class of formulations fordelivering mRNA vaccines in vivo that results in significantly enhanced,and in many respects synergistic, immune responses including enhancedantigen generation and functional antibody production withneutralization capability. These results can be achieved even whensignificantly lower doses of the mRNA are administered in comparisonwith mRNA doses used in other classes of lipid based formulations. Theformulations of the invention have demonstrated significant unexpectedin vivo immune responses sufficient to establish the efficacy offunctional mRNA vaccines as prophylactic and therapeutic agents.Additionally, self-replicating RNA vaccines rely on viral replicationpathways to deliver enough RNA to a cell to produce an immunogenicresponse. The formulations of the invention do not require viralreplication to produce enough protein to result in a strong immuneresponse. Thus, the mRNA of the invention are not self-replicating RNAand do not include components necessary for viral replication.

The invention involves, in some aspects, the surprising finding thatlipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA vaccines, including chemically modified andunmodified mRNA vaccines. The efficacy of mRNA vaccines formulated inLNP was examined in vivo using several distinct antigens. The resultspresented herein demonstrate the unexpected superior efficacy of themRNA vaccines formulated in LNP over other commercially availablevaccines.

In addition to providing an enhanced immune response, the formulationsof the invention generate a more rapid immune response with fewer dosesof antigen than other vaccines tested. The mRNA-LNP formulations of theinvention also produce quantitatively and qualitatively better immuneresponses than vaccines formulated in a different carriers. The datadescribed herein demonstrate that the formulations of the inventionproduced significant unexpected improvements over existing antigenvaccines. Additionally, the mRNA-LNP formulations of the invention aresuperior to other vaccines even when the dose of mRNA is lower thanother vaccines.

The LNP used in the studies described herein has been used previously todeliver siRNA in various animal models as well as in humans. In view ofthe observations made in association with the siRNA delivery of LNPformulations, the fact that LNP is useful in vaccines is quitesurprising. It has been observed that therapeutic delivery of siRNAformulated in LNP causes an undesirable inflammatory response associatedwith a transient IgM response, typically leading to a reduction inantigen production and a compromised immune response. In contrast to thefindings observed with siRNA, the LNP-mRNA formulations of the inventionare demonstrated herein to generate enhanced IgG levels, sufficient forprophylactic and therapeutic methods rather than transient IgMresponses.

Nucleic Acids/Polynucleotides

Human cytomegalovirus (HCMV) vaccines, as provided herein, comprise atleast one (one or more) ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide. Theterm “nucleic acid,” in its broadest sense, includes any compound and/orsubstance that comprises a polymer of nucleotides. These polymers arereferred to as polynucleotides.

In some embodiments, at least one RNA polynucleotide of a HCMV vaccineis encoded by at least one nucleic acid sequence selected from any ofSEQ ID NOs: 1-31, 58, 60, 62, 64, 66 and 68. In some embodiments, atleast one RNA polynucleotide of a HCMV vaccine is encoded by at leastone fragment of a nucleic acid sequence selected from any of SEQ ID NOs:1-31, 58, 60, 62, 64, 66 and 68.

In some embodiments, an RNA vaccine comprises an RNA polynucleotidehaving an open reading frame encoded by SEQ ID NO:58, or an antigenicfragment or epitope thereof; an RNA polynucleotide having an openreading frame encoded by SEQ ID NO:60, or an antigenic fragment orepitope thereof; an RNA polynucleotide having an open reading frameencoded by SEQ ID NO:62, or an antigenic fragment or epitope thereof; anRNA polynucleotide having an open reading frame encoded by SEQ ID NO:64,or an antigenic fragment or epitope thereof; an RNA polynucleotidehaving an open reading frame encoded by SEQ ID NO:66, or an antigenicfragment or epitope thereof, and an RNA polynucleotide having an openreading frame encoded by SEQ ID NO:68, or an antigenic fragment orepitope thereof.

Nucleic acids (also referred to as polynucleotides) may be or mayinclude, for example, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo. In some preferred embodiments, anmRNA is translated in vivo. The skilled artisan will appreciate that,except where otherwise noted, polynucleotide sequences set forth in theinstant application will recite “T”s in a representative DNA sequencebut where the sequence represents RNA (e.g., mRNA), the “T”s would besubstituted for “U”s. Thus, any of the RNA polynucleotides encoded by aDNA identified by a particular sequence identification number may alsocomprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA,where each “T” of the DNA sequence is substituted with “U.”

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics.

Some embodiments of the present disclosure provide HCMV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment or epitope thereof. Some embodiments of thepresent disclosure provide HCMV vaccines that include at least one RNApolynucleotide having an open reading frame encoding two or more HCMVantigenic polypeptides or an immunogenic fragment or epitope thereof.Some embodiments of the present disclosure provide HCMV vaccines thatinclude two or more RNA polynucleotides having an open reading frameencoding two or more HCMV antigenic polypeptides or immunogenicfragments or epitopes thereof. The one or more HCMV antigenicpolypeptides may be encoded on a single RNA polynucleotide or may beencoded individually on multiple (e.g., two or more) RNApolynucleotides.

Some embodiments of the present disclosure provide HCMV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having asingle open reading frame encoding two or more (for example, two, three,four, five, or more) HCMV antigenic polypeptides or an immunogenicfragment or epitope thereof. Some embodiments of the present disclosureprovide HCMV vaccines that include at least one ribonucleic acid (RNA)polynucleotide having more than one open reading frame, for example,two, three, four, five or more open reading frames encoding two, three,four, five or more HCMV antigenic polypeptides. In either of theseembodiments, the at least one RNA polynucleotide may encode two or moreHCMV antigenic polypeptides selected from gH, gB, gL, gO, gM, gN, UL83,UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In someembodiments, the at least one RNA polynucleotide encodes UL83 and UL123.In some embodiments, the at least one RNA polynucleotide encodes gH andgL. In some embodiments, the at least one RNA polynucleotide encodesUL128, UL130, and UL131A. In some embodiments, the at least one RNApolynucleotide encodes gH, gL, UL128, UL130, and UL131A.

In some embodiments, a vaccine comprises an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide gH, or anantigenic fragment or epitope thereof; an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide gL, or anantigenic fragment or epitope thereof, an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide UL128, or anantigenic fragment or epitope thereof; an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide UL130, or anantigenic fragment or epitope thereof; an RNA polynucleotide having anopen reading frame encoding HCMV antigenic polypeptide UL131A, or anantigenic fragment or epitope thereof, and an RNA polynucleotide havingan open reading frame encoding HCMV antigenic polypeptide gB, or anantigenic fragment or epitope thereof.

In some embodiments, in which the at least one RNA polynucleotide has asingle open reading frame encoding two or more (for example, two, three,four, five, or more) HCMV antigenic polypeptides, the RNA polynucleotidemay further comprise additional sequence, for example, a linker sequenceor a sequence that aids in the processing of the HCMV RNA transcripts orpolypeptides, for example a cleavage site sequence. In some embodiments,the additional sequence may be a protease sequence, such as a furinsequence. Furin, also referred to as PACE (paired basic amino acidcleaving enzyme), is a calcium-dependent serine endoprotease thatcleaves precursor proteins into biologically active products at pairedbasic amino acid processing sites. Some of its substrates include thefollowing: proparathyroid hormone, transforming growth factor beta 1precursor, proalbumin, pro-beta-secretase, membrane type-1 matrixmetalloproteinase, beta subunit of pro-nerve growth factor, and vonWillebrand factor. The envelope proteins of certain viruses must becleaved by furin in order to become fully functional, while some virusesrequire furin processing during their entry into host cells. T cellsrequire furin to maintain peripheral immune tolerance. In someembodiments, the additional sequence may be self-cleaving 2A peptide,such as a P2A, E2A, F2A, and T2A sequence. In some embodiments, thelinker sequences and cleavage site sequences are interspersed betweenthe sequences encoding HCMV polypeptides. 2A peptides are“self-cleaving” small peptides, approximately 18-22 amino acids inlength. Ribosomes skip the synthesis of a glycyl-prolyl peptide bond atthe C-terminus of a 2A peptide, resulting in the cleavage of the 2Apeptide and its immediate downstream peptide. They are frequently usedin biomedical research to allow for the simultaneous expression of morethan one gene in cells using a single plasmid. There are a number of 2Apeptides, including the following: foot-and-mouth disease virus (FMDV)2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), porcine teschovirus-12A (P2A), and Thoseaasigna virus 2A (T2A). T2A has the highest cleavageefficiency (close to 100%), followed by E2A, P2A, and F2A. Amino acidsequences are the following: P2A:(GSG)ATNFSLLKQAGDVEENPGP (SEQ IDNO:70); T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO:71); E2A:(GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO:72); F2A:(GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:73). In some embodiments, thelinker sequences and cleavage site sequences are interspersed betweenthe sequences encoding HCMV polypeptides. In some embodiments, the RNApolynucleotide is encoded by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30 or SEQ ID NO: 31.

In some embodiments, a RNA polynucleotide of a HCMV vaccine encodes2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5,3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9,6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antigenic polypeptides. Insome embodiments, a RNA polynucleotide of a HCMV vaccine encodes atleast 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 antigenic polypeptides.In some embodiments, a RNA polynucleotide of a HCMV vaccine encodes atleast 100 or at least 200 antigenic polypeptides. In some embodiments, aRNA polynucleotide of a HCMV vaccine encodes 1-10, 5-15, 10-20, 15-25,20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or 2-100 antigenicpolypeptides.

Polynucleotides of the present disclosure, in some embodiments, arecodon optimized. Codon optimization methods are known in the art and maybe used as provided herein. Codon optimization, in some embodiments, maybe used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art—non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 90%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 85%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 80%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide.

In some embodiments, a codon optimized sequence shares between 65% and85% (e.g., between about 67% and about 85% or between about 67% andabout 80%) sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide. In some embodiments, a codon optimized sequenceshares between 65% and 75 or about 80% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a polypeptide or protein of interest(e.g., an antigenic protein or polypeptide.

The skilled artisan will appreciate that, except where otherwise noted,polynucleotide sequences set forth in the instant application willrecite “T”s in a representative DNA sequence but where the sequencerepresents RNA, the “T”s would be substituted for “U”s.

Antigens/Antigenic Polypeptides

In some embodiments, an antigenic polypeptide is an HCMV glycoprotein.For example, a HCMV glycoprotein may be HCMV gB, gH, gL, gO, gN, or gMor an immunogenic fragment or epitope thereof. In some embodiments, theantigenic polypeptide is a HCMV gH polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gL polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gB polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gO polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gN polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gM polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gC polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gN polypeptide. In some embodiments, theantigenic polypeptide is a HCMV gM polypeptide.

In some embodiments, an antigenic polypeptide is a HCMV protein selectedfrom UL83, UL123, UL128, UL130, and UL131A or an immunogenic fragment orepitope thereof. In some embodiments, the antigenic polypeptide is aHCMV UL83 polypeptide. In some embodiments, the antigenic polypeptide isa HCMV UL123 polypeptide. In some embodiments, the antigenic polypeptideis a HCMV UL128 polypeptide. In some embodiments, the antigenicpolypeptide is a HCMV UL130 polypeptide. In some embodiments, theantigenic polypeptide is a HCMV UL131A polypeptide.

In some embodiments, the antigenic HCMV polypeptide comprises two ormore HCMV polypeptides. The two or more HCMV polypeptides can be encodedby a single RNA polynucleotide or can be encoded by two or more RNApolynucleotides, for example, each glycoprotein encoded by a separateRNA polynucleotide. In some embodiments, the two or more HCMVpolypeptides can be any combination of HCMV gH, gL, gB, gO, gN, gM,UL83, UL123, UL128, UL130, and UL131A polypeptides or immunogenicfragments or epitopes thereof. In some embodiments, the two or more HCMVpolypeptides can be any combination of HCMV gH and a polypeptideselected from gL, gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131Apolypeptides or immunogenic fragments or epitopes thereof. In someembodiments, the two or more HCMV polypeptides can be any combination ofHCMV gB and a polypeptide selected from gH, gL, gO, gN, gM, UL83, UL123,UL128, UL130, and UL131A polypeptides or immunogenic fragments orepitopes thereof. In some embodiments, the two or more HCMV polypeptidescan be any combination of HCMV gL and a polypeptide selected from gH,gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides orimmunogenic fragments or epitopes thereof. In some embodiments, the twoor more HCMV polypeptides can be any combination of HCMV gH, gL and apolypeptide selected from gB, gO, gN, gM, UL83, UL123, UL128, UL130, andUL131A polypeptides or immunogenic fragments or epitopes thereof. Insome embodiments, the two or more HCMV polypeptides can be anycombination of HCMV gH, gL, and a glycoprotein selected from gB, gH, gK,gL, gC, gN, and gM polypeptides or immunogenic fragments or epitopesthereof. In some embodiments, the two or more HCMV polypeptides can beany combination of HCMV gH, gL, and a polypeptide selected from UL83,UL123, UL128, UL130, and UL131A polypeptides or immunogenic fragments orepitopes thereof. In some embodiments, the two or more HCMV polypeptidesare UL128, UL130, and UL131A. In some embodiments, the two or more HCMVpolypeptides are gH and gL. In some embodiments, the two or more HCMVpolypeptides are gH, gL, UL128, UL130, and UL131A. In some embodiments,the two or more HCMV polypeptides are gB, gH, gL, UL128, UL130, andUL131A.

The present disclosure includes variant HCMV antigenic polypeptides. Insome embodiments, the variant HCMV antigenic polypeptide is a variantHCMV gH polypeptide. In some embodiments, the variant HCMV antigenicpolypeptide is a variant HCMV gL polypeptide. In some embodiments, thevariant HCMV antigenic polypeptide is a variant HCMV gB polypeptide. Thevariant HCMV polypeptides are designed to expedite passage of theantigenic polypeptide through the ER/golgi, leading to increased surfaceexpression of the antigen. In some embodiments, the variant HCMVpolypeptides are truncated to delete one or more of the followingdomains: hydrophobic membrane proximal domain, transmembrane domain, andcytoplasmic domain. In some embodiments, the variant HCMV polypeptidesare truncated to include only the ectodomain sequence. For example, thevariant HCMV polypeptide can be a truncated HCMV gH polypeptide,truncated HCMV gB polypeptide, or truncated HCMV gL polypeptidecomprising at least amino acids 1-124, including, for example, aminoacids 1-124, 1-140, 1-160, 1-200, 1-250, 1-300, 1-350, 1-360, 1-400,1-450, 1-500, 1-511, 1-550, and 1-561, as well as polypeptide fragmentshaving fragment sizes within the recited size ranges.

In some embodiments, a HCMV antigenic polypeptide is longer than 25amino acids and shorter than 50 amino acids. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. Polypeptides may also comprise single chain or multichainpolypeptides such as antibodies or insulin and may be associated orlinked. Most commonly, disulfide linkages are found in multichainpolypeptides. The term polypeptide may also apply to amino acid polymersin which at least one amino acid residue is an artificial chemicalanalogue of a corresponding naturally-occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants possessat least 50% identity to a native or reference sequence. In someembodiments, variants share at least 80%, or at least 90% identity witha native or reference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains at least one amino acid thatwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, for example, phenylalanine may act as aninactivating substitution for tyrosine; or alanine may act as aninactivating substitution for serine.

“Orthologs” refers to genes in different species that evolved from acommon ancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution. Identification of orthologs iscritical for reliable prediction of gene function in newly sequencedgenomes.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, for example, substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present disclosure provides several types of compositions that arepolynucleotide or polypeptide based, including variants and derivatives.These include, for example, substitutional, insertional, deletion andcovalent variants and derivatives. The term “derivative” is usedsynonymously with the term “variant” but generally refers to a moleculethat has been modified and/or changed in any way relative to a referencemolecule or starting molecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. For example, sequence tags or amino acids, such as oneor more lysines, can be added to peptide sequences (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidedetection, purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. Substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminior any combination thereof.

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions). As used herein whenreferring to polypeptides the terms “site” as it pertains to amino acidbased embodiments is used synonymously with “amino acid residue” and“amino acid side chain.” As used herein when referring topolynucleotides the terms “site” as it pertains to nucleotide basedembodiments is used synonymously with “nucleotide.” A site represents aposition within a peptide or polypeptide or polynucleotide that may bemodified, manipulated, altered, derivatized or varied within thepolypeptide or polynucleotide based molecules.

As used herein the terms “termini” or “terminus” when referring topolypeptides or polynucleotides refers to an extremity of a polypeptideor polynucleotide respectively. Such extremity is not limited only tothe first or final site of the polypeptide or polynucleotide but mayinclude additional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH2))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N- and C-termini.Alternatively, the termini of the polypeptides may be modified such thatthey begin or end, as the case may be, with a non-polypeptide basedmoiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or greater than 100 amino acids in length. In anotherexample, any protein that includes a stretch of 20, 30, 40, 50, or 100amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%identical to any of the sequences described herein can be utilized inaccordance with the disclosure. In some embodiments, a polypeptideincludes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in anyof the sequences provided or referenced herein.

Polypeptide or polynucleotide molecules of the present disclosure mayshare a certain degree of sequence similarity or identity with thereference molecules (e.g., reference polypeptides or referencepolynucleotides), for example, with art-described molecules (e.g.,engineered or designed molecules or wild-type molecules). The term“identity” as known in the art, refers to a relationship between thesequences of two or more polypeptides or polynucleotides, as determinedby comparing the sequences. In the art, identity also means the degreeof sequence relatedness between them as determined by the number ofmatches between strings of two or more amino acid residues or nucleicacid residues. Identity measures the percent of identical matchesbetween the smaller of two or more sequences with gap alignments (ifany) addressed by a particular mathematical model or computer program(e.g., “algorithms”). Identity of related peptides can be readilycalculated by known methods. “% identity” as it applies to polypeptideor polynucleotide sequences is defined as the percentage of residues(amino acid residues or nucleic acid residues) in the candidate aminoacid or nucleic acid sequence that are identical with the residues inthe amino acid sequence or nucleic acid sequence of a second sequenceafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent identity. Methods and computer programs forthe alignment are well known in the art. It is understood that identitydepends on a calculation of percent identity but may differ in value dueto gaps and penalties introduced in the calculation. Generally, variantsof a particular polynucleotide or polypeptide have at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% but less than 100% sequence identity to thatparticular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402). Another popular local alignmenttechnique is based on the Smith-Waterman algorithm (Smith, T. F. &Waterman, M. S. (1981) “Identification of common molecularsubsequences.” J. Mol. Biol. 147:195-197.) A general global alignmenttechnique based on dynamic programming is the Needleman—Wunsch algorithm(Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable tothe search for similarities in the amino acid sequences of twoproteins.” J. Mol. Biol. 48:443-453.). More recently a Fast OptimalGlobal Sequence Alignment Algorithm (FOGSAA) has been developed thatpurportedly produces global alignment of nucleotide and proteinsequences faster than other optimal global alignment methods, includingthe Needleman—Wunsch algorithm. Other tools are described herein,specifically in the definition of “identity” below.

As used herein, the term “homology” refers to the overall relatednessbetween polymeric molecules, e.g. between nucleic acid molecules (e.g.DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNAmolecules and/or RNA molecules) and/or polypeptide molecules) that sharea threshold level of similarity or identity determined by alignment ofmatching residues are termed homologous. Homology is a qualitative termthat describes a relationship between molecules and can be based uponthe quantitative similarity or identity. Similarity or identity is aquantitative term that defines the degree of sequence match between twocompared sequences. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). Two polynucleotide sequences are consideredhomologous if the polypeptides they encode are at least 50%, 60%, 70%,80%, 90%, 95%, or even 99% for at least one stretch of at least 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Twoprotein sequences are considered homologous if the proteins are at least50%, 60%, 70%, 80%, or 90% identical for at least one stretch of atleast 20 amino acids.

Homology implies that the compared sequences diverged in evolution froma common origin. The term “homolog” refers to a first amino acidsequence or nucleic acid sequence (e.g., gene (DNA or RNA) or proteinsequence) that is related to a second amino acid sequence or nucleicacid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteinsseparated by the event of speciation or to the relationship betweengenes and/or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolvedfrom a common ancestral gene (or protein) by speciation. Typically,orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new functions, even if these are related to theoriginal one.

The term “identity” refers to the overall relatedness between polymericmolecules, for example, between polynucleotide molecules (e.g. DNAmolecules and/or RNA molecules) and/or between polypeptide molecules.Calculation of the percent identity of two polynucleic acid sequences,for example, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Incertain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleic acidsequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleic acid sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleic acid sequencescan, alternatively, be determined using the GAP program in the GCGsoftware package using an NWSgapdna.CMP matrix. Methods commonlyemployed to determine percent identity between sequences include, butare not limited to those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

In some embodiments, the polypeptides further comprise additionalsequences or functional domains. For example, the HCMV polypeptides ofthe present disclosure may comprise one or more linker sequences. Insome embodiments, the HCMV of the present invention may comprise apolypeptide tag, such as an affinity tag (chitin binding protein (CBP),maltose binding protein (MBP), glutathione-S-transferase (GST), SBP-tag,Strep-tag, AviTag, Calmodulin-tag); solubilization tag; chromatographytag (polyanionic amino acid tag, such as FLAG-tag); epitope tag (shortpeptide sequences that bind to high-affinity antibodies, such as V5-tag,Myc-tag, VSV-tag, Xpress tag, E-tag, S-tag, and HA-tag); fluorescencetag (e.g., GFP). In some embodiments, the HCMV of the present inventionmay comprise an amino acid tag, such as one or more lysines, histidines,or glutamates, which can be added to the polypeptide sequences (e.g., atthe N-terminal or C-terminal ends). Lysines can be used to increasepeptide solubility or to allow for biotinylation. Protein and amino acidtags are peptide sequences genetically grafted onto a recombinantprotein. Sequence tags are attached to proteins for various purposes,such as peptide purification, identification, or localization, for usein various applications including, for example, affinity purification,protein array, western blotting, immunofluorescence, andimmunoprecipitation. Such tags are subsequently removable by chemicalagents or by enzymatic means, such as by specific proteolysis or inteinsplicing.

Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support.

Multiprotein and Multicomponent Vaccines

The present disclosure encompasses HCMV vaccines, e.g., vaccines againsthuman cytomegalovirus, comprising multiple RNA (e.g., mRNA)polynucleotides, each encoding a single antigenic polypeptide, as wellas HCMV vaccines comprising a single RNA polynucleotide encoding morethan one antigenic polypeptide (e.g., as a fusion polypeptide). Thus, itshould be understood that a vaccine composition comprising a RNApolynucleotide having an open reading frame encoding a first HCMVantigenic polypeptide and a RNA polynucleotide having an open readingframe encoding a second HCMV antigenic polypeptide encompasses (a)vaccines that comprise a first RNA polynucleotide encoding a first HCMVantigenic polypeptide and a second RNA polynucleotide encoding a secondHCMV antigenic polypeptide, and (b) vaccines that comprise a single RNApolynucleotide encoding a first and second HCMV antigenic polypeptide(e.g., as a fusion polypeptide). HCMV RNA vaccines of the presentdisclosure, in some embodiments, comprise 2-10 (e.g., 2, 3, 4, 5, 6, 7,8, 9 or 10), or more, RNA polynucleotides having an open reading frame,each of which encodes a different HCMV antigenic polypeptide (or asingle RNA polynucleotide encoding 2-10, or more, different HCMVantigenic polypeptides). In some embodiments, an HCMV RNA vaccinecomprises a RNA polynucleotide having an open reading frame encoding anHCMV glycoprotein. In some embodiments, an HCMV RNA vaccine comprises aRNA polynucleotide having an open reading frame encoding an HCMVglycoprotein B (gB), a RNA polynucleotide having an open reading frameencoding an HCMV glycoprotein M (gM), a RNA polynucleotide having anopen reading frame encoding an HCMV glyprotein N (gN), a RNApolynucleotide having an open reading frame encoding an HCMVglycoprotein H (gH), a RNA polynucleotide having an open reading frameencoding an HCMV glycoprotein L (gL), and a RNA polynucleotide having anopen reading frame encoding an HCMV glycoprotein O (gO). In someembodiments, an HCMV RNA vaccine comprises a RNA polynucleotide havingan open reading frame encoding an HCMV gB protein. In some embodiments,an HCMV RNA vaccine comprises a RNA polynucleotide having an openreading frame encoding an HCMV UL128 protein. In some embodiments, anHCMV RNA vaccine comprises a RNA polynucleotide having an open readingframe encoding an HCMV UL130 protein. In some embodiments, an HCMV RNAvaccine comprises a RNA polynucleotide having an open reading frameencoding an HCMV UL131 protein. In some embodiments, an HCMV RNA vaccinecomprises a RNA polynucleotide having an open reading frame encoding anHCMV gM and gN proteins. In some embodiments, an HCMV RNA vaccinecomprises a RNA polynucleotide having an open reading frame encoding anHCMV gH, gL, and gO proteins. In some embodiments, an HCMV RNA vaccinecomprises a RNA polynucleotide having an open reading frame encoding anHCMV gH, gL, UL128, UL130, and UL131A proteins. In some embodiments, anHCMV RNA vaccine comprises RNA polynucleotides having one or more openreading frames encoding an HCMV UL83, UL128, UL123, UL130, or UL131Aprotein. In some embodiments, the HCMV RNA vaccine further comprises aRNA polynucleotide having an open reading frame encoding one or more(e.g., 2, 3, 4, 5, 6 or 7) HCMV proteins.

In some embodiments, an HCMV RNA vaccine comprises RNA polynucleotideshaving one or more open reading frames encoding HCMV gH, gL, UL128,UL130, and UL131A proteins, or fragments thereof, and an HCMV gBprotein, or fragment thereof.

In some embodiments, an HCMV RNA vaccine comprises an RNA polynucleotidehaving an open reading frame encoding a gH protein or a fragmentthereof, an RNA polynucleotide having an open reading frame encoding agL protein or a fragment thereof, an RNA polynucleotide having an openreading frame encoding a UL128 protein or a fragment thereof, an RNApolynucleotide having an open reading frame encoding a UL130 protein ora fragment thereof, an RNA polynucleotide having an open reading frameencoding a UL131A protein or a fragment thereof, and an an RNApolynucleotide having an open reading frame encoding a gB protein, or afragment thereof.

In some embodiments, a RNA polynucleotide encodes an HCMV antigenicpolypeptide fused to a signal peptide (e.g., SEQ ID NO: 53 or 54). Thesignal peptide may be fused at the N-terminus or the C-terminus of theantigenic polypeptide.

Signal Peptides

In some embodiments, antigenic polypeptides encoded by HCMV nucleicacids comprise a signal peptide. Signal peptides, comprising theN-terminal 15-60 amino acids of proteins, are typically needed for thetranslocation across the membrane on the secretory pathway and thusuniversally control the entry of most proteins both in eukaryotes andprokaryotes to the secretory pathway. Signal peptides generally includethree regions: an N-terminal region of differing length, which usuallycomprises positively charged amino acids, a hydrophobic region, and ashort carboxy-terminal peptide region. In eukaryotes, the signal peptideof a nascent precursor protein (pre-protein) directs the ribosome to therough endoplasmic reticulum (ER) membrane and initiates the transport ofthe growing peptide chain across it. The signal peptide is notresponsible for the final destination of the mature protein, however.Secretory proteins devoid of further address tags in their sequence areby default secreted to the external environment. Signal peptides arecleaved from precursor proteins by an endoplasmic reticulum(ER)-resident signal peptidase or they remain uncleaved and function asa membrane anchor. During recent years, a more advanced view of signalpeptides has evolved, showing that the functions and immunodorminance ofcertain signal peptides are much more versatile than previouslyanticipated.

HCMV vaccines of the present disclosure may comprise, for example, RNApolynucleotides encoding an artificial signal peptide, wherein thesignal peptide coding sequence is operably linked to and is in framewith the coding sequence of the HCMV antigenic polypeptide. Thus, HCMVvaccines of the present disclosure, in some embodiments, produce anantigenic polypeptide comprising a HCMV antigenic polypeptide fused to asignal peptide. In some embodiments, a signal peptide is fused to theN-terminus of the HCMV antigenic polypeptide. In some embodiments, asignal peptide is fused to the C-terminus of the HCMV antigenicpolypeptide.

In some embodiments, the signal peptide fused to the HCMV antigenicpolypeptide is an artificial signal peptide. In some embodiments, anartificial signal peptide fused to the HCMV antigenic polypeptideencoded by the HCMV RNA vaccine is obtained from an immunoglobulinprotein, e.g., an IgE signal peptide or an IgG signal peptide. In someembodiments, a signal peptide fused to the HCMV antigenic polypeptideencoded by an HCMV mRNA vaccine is an Ig heavy chain epsilon-1 signalpeptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS (SEQ IDNO: 53). In some embodiments, a signal peptide fused to a HCMV antigenicpolypeptide encoded by the HCMV RNA vaccine is an IgG_(k) chain V-IIIregion HAH signal peptide (IgG_(k) SP) having the sequence ofMETPAQLLFLLLLWLPDTTG (SEQ ID NO: 54). In some embodiments, a signalpeptide fused to the HCMV antigenic polypeptide encoded by an HCMV RNAvaccine has an amino acid sequence set forth in SEQ ID NO: 53 or SEQ IDNO: 54. The examples disclosed herein are not meant to be limiting andany signal peptide that is known in the art to facilitate targeting of aprotein to ER for processing and/or targeting of a protein to the cellmembrane may be used in accordance with the present disclosure.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide may have alength of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55,20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50,30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45,15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30,20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

Non-limiting examples of HCMV antigenic polypeptides fused to signalpeptides, which are encoded by the HCMV RNA vaccine of the presentdisclosure, may be found in Table 2, SEQ ID NOs: 32-52.

A signal peptide is typically cleaved from the nascent polypeptide atthe cleavage junction during ER processing. The mature HCMV antigenicpolypeptide produce by HCMV RNA vaccine of the present disclosuretypically does not comprise a signal peptide.

Chemical Modifications

HCMV RNA vaccines of the present disclosure comprise, in someembodiments, at least one ribonucleic acid (RNA) polynucleotide havingan open reading frame encoding at least one HCMV antigenic polypeptide,or an immunogenic fragment thereof, that comprises at least one chemicalmodification.

The terms “chemical modification” and “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides inat least one of their position, pattern, percent or population.Generally, these terms do not refer to the ribonucleotide modificationsin naturally occurring 5′-terminal mRNA cap moieties. With respect to apolypeptide, the term “modification” refers to a modification relativeto the canonical set 20 amino acids. Polypeptides, as provided herein,are also considered “modified” of they contain amino acid substitutions,insertions or a combination of substitutions and insertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Modifications of polynucleotides include, without limitation, thosedescribed herein. Polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) may comprise modifications that arenaturally-occurring, non-naturally-occurring or the polynucleotide maycomprise a combination of naturally-occurring andnon-naturally-occurring modifications. Polynucleotides may include anyuseful modification, for example, of a sugar, a nucleobase, or aninternucleoside linkage (e.g., to a linking phosphate, to aphosphodiester linkage or to the phosphodiester backbone).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on an internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphodiester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker may be incorporated into polynucleotides of thepresent disclosure.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the vaccines of the presentdisclosure include, but are not limited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6, N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-α-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uridine; N1-ethyl-pseudo-uridine; uridine 5-oxyaceticacid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2(thio)pseudouracil; 2′ deoxyuridine; 2′ fluorouridine; 2-(thio)uracil;2,4-(dithio)psuedouracil; 2′ methyl, 2′amino, 2′azido, 2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP;2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxyuridine; 2′fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridineTP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxyl}propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of pseudouridine (ψ), N1-methylpseudouridine (m¹ψ),N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides) include a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of 1-methyl-pseudouridine (m¹ψ), 5-methoxy-uridine(m⁵U), 5-methyl-cytidine (m⁵C), pseudouridine (ψ), α-thio-guanosine andα-thio-adenosine. In some embodiments, polynucleotides includes acombination of at least two (e.g., 2, 3, 4 or more) of theaforementioned modified nucleobases.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise pseudouridine (w) and 5-methyl-cytidine(m⁵C). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ). Insome embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine(s²U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2-thiouridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine(mo⁵U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo⁵U) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyluridine. In some embodiments polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A). In some embodiments, polynucleotides (e.g.,RNA polynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m⁵C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m⁵C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Exemplary nucleobases and In some embodiments, a modified nucleobase isa modified cytosine. nucleosides having a modified uridine include5-cyano uridine, and 4′-thio uridine. In some embodiments, a modifiednucleobase is a modified adenine. Exemplary nucleobases and nucleosideshaving a modified adenine include 7-deaza-adenine, 1-methyl-adenosine(m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

In some embodiments, a modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof (e.g., in the mRNA including or excluding the polyAtail). In some embodiments, all nucleotides X in a polynucleotide of thepresent disclosure (or in a given sequence region thereof) are modifiednucleotides, wherein X may any one of nucleotides A, G, U, C, or any oneof the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C orA+G+C.

The polynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the polynucleotides may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the polynucleotide is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe polynucleotide is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

In some embodiments a codon optimized RNA may, for instance, be one inwhich the levels of G/C are enhanced. The G/C-content of nucleic acidmolecules may influence the stability of the RNA. RNA having anincreased amount of guanine (G) and/or cytosine (C) residues may befunctionally more stable than nucleic acids containing a large amount ofadenine (A) and thymine (T) or uracil (U) nucleotides. WO02/098443discloses a pharmaceutical composition containing an mRNA stabilized bysequence modifications in the translated region. Due to the degeneracyof the genetic code, the modifications work by substituting existingcodons for those that promote greater RNA stability without changing theresulting amino acid. The approach is limited to coding regions of theRNA.

Thus, in some embodiments, the RNA (e.g., mRNA) vaccines comprise a5′UTR element, an optionally codon optimized open reading frame, and a3′UTR element, a poly(A) sequence and/or a polyadenylation signalwherein the RNA is not chemically modified. In some embodiments, themodified nucleobase is a modified uracil. Exemplary nucleobases andnucleosides having a modified uracil include pseudouridine (w),pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine,2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U),4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (hoSU),5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (moSU),uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester(mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U),5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U),5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine(mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U),5-methylaminomethyl-2-seleno-uridine (mnm5se2U),5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine(cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm5U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm5s2U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ),5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3w),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,N1-ethyl-pseudouridine 3-(3-amino-3-carboxypropyl)uridine (acp3U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ),5-(isopentenylaminomethyl)uridine (inm5U),5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um),3,2′-O-dimethyl-uridine (m3Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C),N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m5Cm), N4-acetyl-2′-O-methyl-cytidine (ac4Cm),N4,2′-O-dimethyl-cytidine (m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm),N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (ml A),2-methyl-adenine (m2A), N6-methyl-adenosine (m6A),2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine(i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A),N6-(cis-hydroxyisopentenyl)adenosine (io6A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A),N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine(t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A),N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A),N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl-adenosine(m62Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G),N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m2Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm),1-methyl-2′-O-methyl-guanosine (m1Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m1Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In Vitro Transcription of RNA (e.g., mRNA)

HCMV vaccines of the present disclosure comprise at least one RNApolynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example,is transcribed in vitro from template DNA, referred to as an “in vitrotranscription template.” In some embodiments, an in vitro transcriptiontemplate encodes a 5′ untranslated (UTR) region, contains an openreading frame, and encodes a 3′ UTR and a polyA tail. The particularnucleic acid sequence composition and length of an in vitrotranscription template will depend on the mRNA encoded by the template.

A “5′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides).

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of HCMV inhumans and other mammals. HCMV RNA vaccines can be used as therapeuticor prophylactic agents. They may be used in medicine to prevent and/ortreat infectious disease. In exemplary aspects, the HCMV RNA vaccines ofthe invention are used to provide prophylactic protection from humancytomegalovirus infection and may be particularly useful for preventionand/or treatment of immunocompromised and infant patients to prevent orto reduce the severity and/or duration of the clinical manifestation ofthe cytomegalovirus infection. In some embodiments, vaccines describedherein reduce or prevent congenital transmission of HCMV from mother tochild.

Broad Spectrum Vaccines

HCMV RNA (e.g., mRNA) vaccines can be used as therapeutic orprophylactic agents. It is envisioned that there may be situations wherepersons are at risk for infection with more than one betacoronovirus,for example, at risk for infection with HCMV. RNA (e.g., mRNA)therapeutic vaccines are particularly amenable to combinationvaccination approaches due to a number of factors including, but notlimited to, speed of manufacture, ability to rapidly tailor vaccines toaccommodate perceived geographical threat, and the like. Moreover,because the vaccines utilize the human body to produce the antigenicprotein, the vaccines are amenable to the production of larger, morecomplex antigenic proteins, allowing for proper folding, surfaceexpression, antigen presentation, etc. in the human subject. To protectagainst more than one HCMV strain, a combination vaccine can beadministered that includes RNA encoding at least one antigenicpolypeptide of a first HCMV and further includes RNA encoding at leastone antigenic polypeptide of a second HCMV. RNAs (mRNAs) can beco-formulated, for example, in a single LNP or can be formulated inseparate LNPs destined for co-administration.

A method of eliciting an immune response in a subject against a HCMV isprovided in aspects of the invention. The method involves administeringto the subject a HCMV RNA vaccine comprising at least one RNApolynucleotide having an open reading frame encoding at least one HCMVantigenic polypeptide or an immunogenic fragment thereof, therebyinducing in the subject an immune response specific to HCMV antigenicpolypeptide or an immunogenic fragment thereof, wherein anti-antigenicpolypeptide antibody titer in the subject is increased followingvaccination relative to anti-antigenic polypeptide antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the HCMV. An “anti-antigenic polypeptideantibody” is a serum antibody the binds specifically to the antigenicpolypeptide.

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the mRNA vaccines of theinvention. For instance, a traditional vaccine includes but is notlimited to live microorganism vaccines, killed microorganism vaccines,subunit vaccines, protein antigen vaccines, DNA vaccines, etc. Inexemplary embodiments, a traditional vaccine is a vaccine that hasachieved regulatory approval and/or is registered by a national drugregulatory body, for example the Food and Drug Administration (FDA) inthe United States or the European Medicines Agency (EMA).

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log to 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 2 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 3 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 5 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theHCMV.

A method of eliciting an immune response in a subject against a HCMV isprovided in other aspects of the invention. The method involvesadministering to the subject a HCMV RNA vaccine comprising at least oneRNA polynucleotide having an open reading frame encoding at least oneHCMV antigenic polypeptide or an immunogenic fragment thereof, therebyinducing in the subject an immune response specific to HCMV antigenicpolypeptide or an immunogenic fragment thereof, wherein the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine against the HCMV at 2 times to 100times the dosage level relative to the RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine attwice the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine atthree times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at4 times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at5 times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at50 times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times the dosage level relative to the HCMV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times to 1000 times the dosage level relative to the HCMV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times to 1000 times the dosage level relative to the HCMV RNAvaccine.

In other embodiments the immune response is assessed by determininganti-antigenic polypeptide antibody titer in the subject.

In other aspects the invention is a method of eliciting an immuneresponse in a subject against a HCMV by administering to the subject aHCMV RNA vaccine comprising at least one RNA polynucleotide having anopen reading frame encoding at least one HCMV antigenic polypeptide oran immunogenic fragment thereof, thereby inducing in the subject animmune response specific to HCMV antigenic polypeptide or an immunogenicfragment thereof, wherein the immune response in the subject is induced2 days to 10 weeks earlier relative to an immune response induced in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the HCMV. In some embodiments the immuneresponse in the subject is induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine at 2 times to100 times the dosage level relative to the RNA vaccine.

In some embodiments the immune response in the subject is induced 2 daysearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 3 daysearlier relative to an immune response induced in a subject vaccinated aprophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 1 weekearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 2weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 3weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 5weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 10weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

A method of eliciting an immune response in a subject against a HCMV byadministering to the subject a HCMV RNA vaccine having an open readingframe encoding a first antigenic polypeptide, wherein the RNApolynucleotide does not include a stabilization element, and wherein anadjuvant is not coformulated or co-administered with the vaccine is alsoprovided herein.

Standard of Care for CMV Prevention and Treatment

A variety of approaches to preventing and/or treating CMV, includingimmunization strategies, have previously been pursued or are currentlybeing pursued, some of which are summarized below. However, all of theseapproaches have drawbacks and limitations. (Schleiss et al. (2008), CurrTop Microbiol Immunol. 325:361-382).

Ganciclovir and Valganciclovir

In some embodiments, Ganciclovir or Valganciclovir is the standard ofcare therapy for treatment or prevention of CMV infections (Reusser P.et al. (2000); 130(4):101-12; Biron et al. (2006) Antiviral Research71:154-163).

Ganciclovir (marketed as CYTOVENE® and ZIRGAN®) and Valganciclovir (aprodrug form of Ganciclovir marketed as VALCYTE®) are antiviralmedications developed by Hoffmann-La Roche to treat CMV infection. Theyare analogues of 2′-deoxy-guanosine, which competitively inhibits dGTPincorporation into DNA and, in turn, viral replication (Sugawara M etal., J Pharm Sci. 2000; 89(6):781-9). CYTOVENE-IV (ganciclovir sodiumfor injection) is FDA approved “for use only in the treatment ofcytomegalovirus (CMV) retinitis in immunocompromised patients and forthe prevention of CMV disease in transplant patients at risk for CMVdisease.” (FDA Label, 1/31/2006, page 1.)

The recommended dose regimen for CYTOVENE-IV for treatment of CMVretinitis for patients with normal renal function includes an inductionphase of 5 mg/kg (administered intravenously over an hour) every 12hours for 14-21 days, followed by a maintenance phase of 5 mg/kg(administered intravenously over an hour) once daily seven days a weekor 6 mg/kg once daily five days a week. (Id., page 22.) For preventionof CMV in transplant patients with normal renal function, therecommended dose regimen includes 5 mg/kg (administered intravenouslyover an hour) every 12 hours for 7-14 days; then 5 mg/kg once dailyseven days a week or 6 mg/kg once daily five days a week. (Id.)

In a study involving heart transplant patients, at 120 dayspost-transplant, the incidence of CMV in seropositive subjects was 9% insubjects receiving treatment compared to 46% in subjects receiving aplacebo. (Biron et al. (2006) Antiviral Research 71:154-163, page 157.)In a study involving bone marrow transplant subjects, at 100 dayspost-transplant the incidence of CMV in treated subjects was 3% comparedto 43% in subjects treated with a placebo. (Id.)

One form of Ganciclovir that is marketed by Bausch and Lomb, ZIRGAN®, isin the form of an ophthalmic gel, which is FDA approved for treatment ofacute herpetic keratitis (dendritic ulcers.) (FDA label, 9/15/2009, page4; Wilhelmus K R et al., 2010, Cochrane Database Syst Rev 12: CD002898).

VALCYTE® (valganciclovir hydrochloride) in tablet form is FDA approvedin adult patients for treatment of CMV retinitis in patients withacquired immunodeficiency syndrome (AIDS) and prevention of CMV diseasein kidney, heart, and kidney-pancreas transplant patients at high risk.(FDA label, 4/23/2015, page 1.) The dose regimen for VALCYTE® is shownin the following table, as depicted on the FDA label dated Apr. 23,2015:

TABLE 1 Dose regimen for VALCYTE ® DOSAGE AND ADMINISTRATION AdultDosage (2.2) Treatment of CMV Induction: 900 mg (two 450 mg tablets)twice a retinitis day for 21 days Maintenance: 900 mg (two 450 mgtablets) once a day Prevention of CMV 900 mg (two 450 mg tablets) once aday within 10 disease in heart or days of transplantation until 100 dayskidney-pancreas post-transplantation transplant patients Prevention ofCMV 900 mg (two 450 mg tablets) once a day within 10 disease in kidneydays of tansplantation until 200 days transplant patientspost-transplantation Pediatric Dosage (2.3) Prevention of CMV Dose oncea day within 10 days of transplantation disease in kidney until 200 dayspost-transplantation according to transplant patients 4 dosage algorithm(note the calculation of months to 16 years creatinine clearance using amodified Schwartz of age formula in children) Prevention of CMV Doseonce a day within 10 days of transplantation disease in heart until 100days post-transplantation according to transplant patients 1 dosagealgorithm (note the calculation of month to 16 years of creatinineclearance using a modified Schwartz age formula in children)

An oral form of Ganciclovir was found to have low bioavailability.(Biron et al. (2006) Antiviral Research 71:154-163.) Valganciclovir wasreported to have better bioavailability than Ganciclovir. (Pescovitz M Det al., Antimicrob Agents Chemother. 2000; 44(10):2811-5; Biron et al.(2006) Antiviral Research 71:154-163.)

Adverse side effects associated with Ganciclovir and Valganciclovirinclude: fever, rash, diarrhea, and hematologic effects (such asneutropenia, anemia, and thrombocytopenia), as well as potentialreproductive toxicity. Ganciclovir was also found to affect fertilityand to be carcinogenic and teratogenic in animal studies. (Biron et al.(2006) Antiviral Research 71:154-163.)

Phase 3 clinical trials involving treatment of CMV infection withGanciclovir or Valganciclovir include trials associated withClinicalTrials government database identifier numbers: NCT00000143,NCT00000136, NCT00000134, NCT00497796, NCT00227370, NCT00466817, andNCT00294515. Results of clinical trials involving Ganciclovir orValganciclovir are summarized in Biron et al. (2006) Antiviral Research71:154-163, incorporated by reference herein in its entirety.

Experimental vaccines in development for CMV

TransVax™ (Also Known as ASP0113 and VCL-CB01)

TransVax™ is a CMV vaccine being developed by Vical Incorporated andAstellas Pharma Inc. (Smith et al. (2013) Vaccines 1(4):398-414.)TransVax™ is a bivalent DNA vaccine containing plasmids encoding CMVpp65 and gB antigens formulated in CRL1005 poloxamer and benzalkonium.(Id.; Kharfan-Dabaja et al. (2012) Lancet Infect Dis 12:290-99). Thepp65 antigen induces cytotoxic T cell response, conferring cellularimmunity, while the gB antigen elicits both cellular immunity andantigen-specific antibody production. Accordingly, the vaccine isintended to induce both cellular and humoral immune responses. The pp65and gB sequences are modified from wild type protein sequences throughdeletions and codon optimization, as described on pages 402-403 of Smithet al. (2013) Vaccines 1(4):398-414, incorporated by reference herein inits entirety.

TransVax™ has received orphan drug designation in the United States andEurope for hematopoietic stem cell transplantation (HSCT), e.g., bonemarrow transplantation, and solid organ transplantation (SOT) patients.

In a Phase 1 clinical trial, 37.5% and 50% of CMV⁻ subjects, who weredosed with 1 mg and 5 mg, respectively, of the vaccine, demonstratedantibody or T-cell responses. (Page 406 of Smith et al. (2013) Vaccines1(4):398-414.) A Phase 2 clinical trial was conducted in patientsundergoing allogenic haemopoietic stem cell transplantation(ClinicalTrials government database identifier number NCT00285259)(Kharfan-Dabaja et al. (2012) Lancet Infect Dis 12:290-99). Transplantpatients received the experimental vaccine four times, including oncebefore the transplantation. (Id., page 292.) The dose beforetransplantation was administered between 3-5 days beforetransplantation, while the doses after transplantation were administeredbetween 21-42 days after transplantation, and at 84 and 196 days aftertransplantation. (Id.) Endpoints included assessment of safety andreduction in cytomegalovirus viraemia. (Id.) The incidence ofcytomegalovirus viraemia was found to be lower in patients who receivedthe vaccine compared to placebo (32.5% (vaccine group) compared to 61.8%(placebo); Table 2, on page 294 of Kharfan-Dabaja et al.). The vaccinewas also reported to be well-tolerated and safe. (Id., page 295.)However, after vaccine treatment, rates of viraemia necessitationanti-viral treatment resembled those of placebo controls. (Id., page296.)

TransVax™ is currently being tested in a Phase 3 clinical trial fortreatment of hematopoietic cell transplant (HCT) patients, accordedClinicalTrials government database identifier number NCT01877655. Theendpoint for the trial is mortality and end organ disease (EOD) 1 yearafter transplant. The estimated enrollment is 500 and the vaccine isadministered by intramuscular injection. TransVax™ is also currentlybeing tested in a Phase 2 clinical trial in CMV-Seronegative kidneytransplant recipients receiving an organ from a CMV-Seropositive donor,accorded ClinicalTrials government database identifier numberNCT01974206. The primary outcome being measured in this trial isincidence of CMV viremia one year after first administration of thedrug. The enrollment is 150 and the vaccine is administered byintramuscular injection. Subjects included in the trial also receivedganciclovir or valganciclovir from within ten days up transplant throughrandomization.

Clinical trials involving TransVax™ are found at the ClinicalTrialsgovernment website with the following ClinicalTrials government databaseidentifier numbers: NCT02103426, NCT01877655, NCT01974206, andNCT01903928.

US patents and published applications that are assigned to Vical Inc.and relate to CMV include: U.S. Pat. Nos. 8,673,317, 9,180,162,8,278,093, 7,888,112, 7,410,795, which are incorporated by referenceherein in their entireties.

Experimental Vaccines in Development by City of Hope/National CancerInstitute/Helocyte

Several experimental CMV vaccines are being developed by City of Hopeand its licensee Helocyte. US patents and published applications thatare assigned to City of Hope and relate to CMV include: U.S. Pat. Nos.7,387,782, 7,025,969, 6,133,433, 6,207,161, 6,074,645, 6,251,399,6,727,093, 6,726,910, 6,843,992, 6,544,521, 6,951,651, 8,580,276,7,163,685, 6,242,567, 6,835,383, 6,156,317, 6,562,345, US 2014-0065181and US 2015-0216965, which are incorporated by reference herein in theirentireties.

CMVPepVax

CMVPepVax is an experimental vaccine being developed by City of HopeMedical Center, National Cancer Institute, and Helocyte, Inc. Thevaccine includes a pp65 T-cell epitope and a tetanus T-helper epitope inthe form of a chimeric peptide, and also includes the adjuvantPF03512676. (Nakamura R et al., Lancet Heamatology (2016) February;3(2):e87-98). CMVPepVax was tested in a Phase 1b clinical trial onCMV-seropositive patients who were undergoing haemopoietic stem-celltransplantation (HCT). (Id.) The vaccine was administered on days 28 and56 through subcutaneous administration. (Id.) It was reported thatpatients receiving the vaccine showed improved relapse-free survival.(Id.) This clinical trial was accorded ClinicalTrials governmentdatabase identifier number NCT01588015. CMVPepVax is currently beingtested in a Phase 2 clinical trial to measure efficacy in reducing thefrequency of Cytomegalovirus events in patients with hematologicmalignancies undergoing donor stem cell transplant, accordedClinicalTrials government database identifier number NCT02396134.

CMV-MVA Triplex

CMV-MVA-Triplex is an experimental CMV vaccine being developed by Cityof Hope Medical Center, National Cancer Institute, and Helocyte, Inc.(formerly DiaVax Biosciences). This vaccine consists of an inactivatedModified Vaccinia Ankara (MVA) viral vector that encodes the CMVantigens UL83 (pp65), UL123 (IE1) and UL122 (IE2). (NCI DrugDictionary.)

CMV-MVA Triplex is currently being tested in a Phase 2 clinical trialinvestigating efficacy in reducing CMV complications in patientspreviously infected with CMV and undergoing donor hematopoietic celltransplant. This trial has been accorded ClinicalTrials governmentdatabase identifier number NCT02506933. A Phase 1 clinical trial inhealthy volunteers with or without previous exposure to CMV is alsoongoing (ClinicalTrials government database identifier No. NCT01941056).

Pentamer

City of Hope and Helocyte, Inc. are also pursuing a pentameric vaccineusing a Modified Vaccinia Ankara (MVA) viral vector that encodes thefive CMV pentameric subunits. This vaccine is still in preclinicaldevelopment. (Wussow et al. (2014) PLoS Pathog 10(11): e1004524. doi:10.1371/journal.ppat.1004524).

gB/MF59

This experimental vaccine, originally developed in the 1990s combinesthe gB antigen with the MF59 adjuvant. (Pass et al. (2009) J Clin Virol46(Suppl 4):S73-S76.) Several clinical trials that were conducted in the1990s, sponsored by Chiron Corporation, indicated that the vaccine wassafe. (Id., page 2.) Sanofi Pasteur later obtained the rights to thisvaccine. (Id.)

A Phase 2 clinical trial was conducted in postpartum females starting in1999 (with enrollment completed in 2006) using the endpoint of time toCMV infection. (Id., page 3.) Subjects were administered the vaccine at0, 1, and 6 months. (Rieder et al. (2014) Clin Microbiol Infect 20(Suppl. 5):95-102, page 98). Infection with CMV was diagnosed in 8% ofvaccine-treated subjects compared to 14% of placebo-treated subjects,respectively (corresponding to 43% efficacy). Results indicated a 50%reduction in rate of CMV infection in subjects treated with the vaccine(3.3% in test subjects compared to 6.6% in placebo-treated subjects).(Id.; Pass et al. (2009) J Clin Virol 46(Suppl 4):S73-S76., page 4.).The 50% reduction in rate of CMV infection has been described as “lowerthan wished for from a clinical perspective.” (Rieder et al. (2014) ClinMicrobiol Infect 20 (Suppl. 5):95-102, page 98.)

A Phase 2 clinical trial has also been conducted with gB/MF59 in kidneyand liver transplant patients. (Id., page 100.) It was reported that“high gB-antibody titres correlated with shorter duration of viraemia”and that “duration of viraemia and number of days of ganciclovirtreatment were reduced.” (Id.)

Clinical trials involving gB/MF59 are found at the ClinicalTrialsgovernment website with the following ClinicalTrials government databaseidentifier numbers: NCT00133497, NCT00815165, and NCT00125502.

US 2009-0104227, assigned to Sanofi Pasteur SA, is incorporated byreference herein in its entirety.

gB/AS01

GlaxoSmithKline is developing an experimental vaccine that includes thegB antigen combined with the ASO1 adjuvant. (McVoy (2013) ClinicalInfectious Diseases 57(54):S196-9, page S197.) This vaccine is referredto as GSK1492903A. Clinical trials involving GSK1492903A are found atthe ClinicalTrials government website with the following ClinicalTrialsgovernment database identifier numbers: NCT00435396 and NCT01357915.

WO 2016/067239 and WO 2015/181142, filed by GlaxoSmithKline BiologicalsSA, are incorporated by reference herein in their entireties.

Towne Vaccine

The CMV Towne vaccine is a live attenuated vaccine. (McVoy (2013)Clinical Infectious Diseases 57(S4):S196-9, page S197.) This vaccine wasnot successful in protecting against primary maternal infection, atleast when administered at a low dose. (Id.) In a trial involving kidneytransplant subjects, treatment with this vaccine resulted in reductionof severe disease, while only having a minimal impact on mild disease.(Plotkin et al. (1994) Transplantation 58(11):1176-8.)

Live attenuated vaccines in which sections of the Towne genome have beenreplaced with sequence from other “low-passage” strains have also beendeveloped, referred to as “Towne-Toledo chimeras,” which were found tobe well-tolerated in a Phase 1 clinical trial. (McVoy (2013) ClinicalInfectious Diseases 57(S4):S196-9, page S197; Heineman et al. (2006) TheJournal of Infectious Diseases 193:1350-60.) Chimeric viral genomesincluding portions of the Towne genome are described in and incorporatedby reference from U.S. Pat. No. 7,204,990, incorporated by referenceherein in its entirety.

Another approach that is being explored involves co-administering theTowne vaccine with the adjuvant recombinant interleukin-12 (rhIL-12)(Jacobson et al. (2006) Vaccine 24:5311-9.)

CMV-CTL

CMV Targeted T-Cell Program (CMV-CTL) represents a cellularimmunotherapy approach being developed by Atara Biotherapeutics.

A Phase 1 clinical trial used CMV pp65 or pp65/IE1 peptide mixes topulse monocytes to expand CMV CTL and investigated the immunologiceffects. (Bao et al. (2012) J Immunother 35(3):293-298). CMV specificimmune responses were observed in approximately 70% of subjectsreceiving CTL. (Id., page 5.)

A Phase 2 clinical trial is currently ongoing, investigating third partydonor derived CMVpp65 specific T-cells for the treatment of CMVinfection or persistent CMV viremia after allogeneic hematopoietic stemcell transplantation. This trial was assigned ClinicalTrials governmentdatabase identifier number NCT02136797. A second Phase 2 clinical trialis also ongoing, investigating primary transplant donor derived CMVpp65specific T-cells for the treatment of CMV infection or persistent CMVviremia after allogeneic hematopoietic stem cell transplantation. Thistrial was assigned ClinicalTrials government database identifier numberNCT01646645.

Monoclonal Abs Novartis

CSJ148, being developed by Novartis, represents a combination of twomonoclonal antibodies that target gB and the CMV pentameric complex.(Dole et al. (2016) Antimicrob Agents Chemother. April 22;60(5):2881-7). The two antibodies are known as LJP538 and LJP539. (Id.)LJP538, LJP539, and CSJ148 were found to be safe when administeredintravenously to healthy volunteers and revealed expectedpharmacokinetics for IgG. (Id.) CSJ148 is currently in a Phase 2clinical trial investigating efficacy and safety in stem cell transplantpatients (ClinicalTrials government database identifier numberNCT02268526).

Theraclone

TCN-202 is a fully human monoclonal antibody being developed byTheraclone for treatment of CMV infection. TCN-202 was found to be safeand well-tolerated in a Phase 1 clinical trial (ClinicalTrialsgovernment database identifier number NCT01594437). A Phase 2 study wasinitiated in 2013 to investigate efficacy in kidney transplantrecipients. (Theraclone Press Release, Sep. 10, 2013.)

Brincidofovir

Brincidofovir (CMX001) is an experimental lipid-nucleotide conjugatebeing developed by Chimerix, Durham, N.C., for treatment of DNA virusesincluding CMV. Brincidofovir received Fast Track designation from theFDA for CMV.

Results from a Phase 3 clinical trial (called “SUPPRESS”) investigatingprevention of CMV in subjects undergoing hematopoietic celltransplantation (HCT) were announced in February, 2016. (Chimerix PressRelease, Feb. 20, 2016.) It was reported that the trial failed to meetits primary endpoint of preventing CMV at week 24, although ananti-viral effect was observed during the treatment phase. (Id.) Thetrial involved 452 subjects undergoing HCT who were administeredBrincidofovir twice a week for up to fourteen weeks. (Id.) It wasspeculated that increased use of immunosteroids, such ascorticosteroids, for treatment of graft versus host disease (GVHD),after treatment with Brincidofovir, may have contributed to failure toreach the primary endpoint of the trial. (Id.) Other Phase 3 trials wereterminated based on the results of the SUPPRESS trial, but Chimerix hasindicated that they intend to pursue further Phase 2 trials in subjectsundergoing kidney transplants. (Id.)

Information about clinical trials associated with Brincidofovir arefound at the ClinicalTrials government website, including identifiernumbers: NCT02087306, NCT02271347, NCT02167685, NCT02596997,NCT02439970, NCT00793598, NCT01769170, NCT00780182, NCT01241344,NCT00942305, NCT02420080, NCT02439957, NCT01143181, and NCT01610765.

V160

V160 is an experimental CMV vaccine being developed by Merck, which isbased on the attenuated AD169 strain. V160 is currently being tested ina Phase 1 clinical trial evaluating a three dose regimen testing severalformulations in healthy adults. This trial was assigned theClinicalTrials government database identifier number NCT01986010.

Merck is also pursuing vaccines that target the CMV pentameric complex.(Loughney et al. (2015) jbc.M115.652230.) US patents and publishedapplications assigned to Merck Sharp & Dohme Corp include: US2014-0220062 and US 2015-0307850, which are incorporated by referenceherein in their entireties.

Letermovir

Letermovir (AIC246) is an antiviral drug being developed by Merck forthe treatment of CMV infections (Chemaly et al. (2014) New EnglandJournal of Medicine, 370; 19, May 8, 2014, Verghese et al. (2013) DrugsFuture. May; 38(5): 291-298). It was tested in a Phase IIb clinicaltrial investigating prevention of CMV in HSCT recipients, correspondingto ClinicalTrials government database identifier number NCT01063829, andwas found to reduce the incidence of CMV infection in transplantsubjects.

Redvax GmbH/Pfizer

A preclinical candidate targeting CMV was developed by Redvax GmbH,which spun out from Redbiotec AG. This candidate is now being pursued byPfizer Inc.

Patents and patent publications assigned to Redvax GmbH or Pfizer andrelated to CMV include: US 2015-0322115, WO 2015/170287, US2015-0359879, and WO 2014/068001, incorporated by reference herein intheir entireties.

Therapeutic and Prophylactic Compositions

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention, treatment or diagnosis ofHCMV in humans. HCMV RNA vaccines can be used as therapeutic orprophylactic agents. They may be used in medicine to prevent and/ortreat infectious disease. In some embodiments, the HCMV vaccines of theinvention can be envisioned for use in the priming of immune effectorcells, for example, to activate peripheral blood mononuclear cells(PBMCs) ex vivo, which are then infused (re-infused) into a subject.

In exemplary embodiments, a HCMV vaccine containing RNA polynucleotidesas described herein can be administered to a subject (e.g., a mammaliansubject, such as a human subject), and the RNA polynucleotides aretranslated in vivo to produce an antigenic polypeptide. In someembodiments, the subject is a woman of child-bearing age. In someembodiments, vaccines described herein reduce or prevent congenitaltransmission of HCMV from a mother to a child. (Pass et al. (2014) J PedInfect Dis 3 (suppl 1): S2-S6.)

The HCMV RNA vaccines may be induced for translation of a polypeptide(e.g., antigen or immunogen) in a cell, tissue or organism. In exemplaryembodiments, such translation occurs in vivo, although there can beenvisioned embodiments where such translation occurs ex vivo, in cultureor in vitro. In exemplary embodiments, the cell, tissue or organism iscontacted with an effective amount of a composition containing a HCMVRNA vaccine that contains a polynucleotide that has at least one atranslatable region encoding an antigenic polypeptide.

An “effective amount” of the HCMV RNA vaccine is provided based, atleast in part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleosides) and other components of theHCMV RNA vaccine, and other determinants. In general, an effectiveamount of the HCMV RNA vaccine composition provides an induced orboosted immune response as a function of antigen production in the cell,preferably more efficient than a composition containing a correspondingunmodified polynucleotide encoding the same antigen or a peptideantigen. Increased antigen production may be demonstrated by increasedcell transfection (the percentage of cells transfected with the RNAvaccine), increased protein translation from the polynucleotide,decreased nucleic acid degradation (as demonstrated, for example, byincreased duration of protein translation from a modifiedpolynucleotide), or altered antigen specific immune response of the hostcell.

In some embodiments, RNA vaccines (including polynucleotides theirencoded polypeptides) in accordance with the present disclosure may beused for treatment of HCMV. HCMV RNA vaccines may be administeredprophylactically or therapeutically as part of an active immunizationscheme to healthy individuals or early in infection during theincubation phase or during active infection after onset of symptoms. Insome embodiments, the amount of RNA vaccines of the present disclosureprovided to a cell, a tissue or a subject may be an amount effective forimmune prophylaxis.

HCMV RNA vaccines may be administrated with other prophylactic ortherapeutic compounds. As a non-limiting example, a prophylactic ortherapeutic compound may be an adjuvant or a booster. As used herein,when referring to a prophylactic composition, such as a vaccine, theterm “booster” refers to an extra administration of the prophylactic(vaccine) composition. A booster (or booster vaccine) may be given afteran earlier administration of the prophylactic composition. The time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years,11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80years, 85 years, 90 years, 95 years or more than 99 years. In exemplaryembodiments, the time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 6 months or 1 year.

In some embodiments, HCMV RNA vaccines may be administeredintramuscularly or intradermally, similarly to the administration ofinactivated vaccines known in the art. The HCMV RNA vaccines may beutilized in various settings depending on the prevalence of theinfection or the degree or level of unmet medical need. As anon-limiting example, the RNA vaccines may be utilized to treat and/orprevent a variety of infectious disease. RNA vaccines have superiorproperties in that they produce much larger antibody titers and produceresponses early than commercially available anti-virals.

Provided herein are pharmaceutical compositions including HCMV RNAvaccines and RNA vaccine compositions and/or complexes optionally incombination with one or more pharmaceutically acceptable excipients.

HCMV RNA vaccines may be formulated or administered alone or inconjunction with one or more other components. For instance, HCMV RNAvaccines (vaccine compositions) may comprise other components including,but not limited to, adjuvants. In some embodiments, HCMV RNA vaccines donot include an adjuvant (they are adjuvant free).

HCMV RNA vaccines may be formulated or administered in combination withone or more pharmaceutically-acceptable excipients. In some embodiments,vaccine compositions comprise at least one additional active substances,such as, for example, a therapeutically-active substance, aprophylactically-active substance, or a combination of both. Vaccinecompositions may be sterile, pyrogen-free or both sterile andpyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents, such as vaccine compositions, maybe found, for example, in Remington: The Science and Practice ofPharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, HCMV RNA vaccines are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the RNA vaccines or thepolynucleotides contained therein, for example, RNA polynucleotides(e.g., mRNA polynucleotides) encoding antigenic polypeptides.Formulations of the vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

HCMV RNA vaccines can be formulated using one or more excipients to: (1)increase stability; (2) increase cell transfection; (3) permit thesustained or delayed release (e.g., from a depot formulation); (4) alterthe biodistribution (e.g., target to specific tissues or cell types);(5) increase the translation of encoded protein in vivo; and/or (6)alter the release profile of encoded protein (antigen) in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients can include, withoutlimitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with HCMV RNA vaccines (e.g., for transplantation into asubject), hyaluronidase, nanoparticle mimics and combinations thereof.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA vaccine may include one or more stabilizingelements. Stabilizing elements may include for instance a histonestem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has beenidentified. It is associated with the histone stem-loop at the 3′-end ofthe histone messages in both the nucleus and the cytoplasm. Itsexpression level is regulated by the cell cycle; it is peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA vaccines include a coding region, at leastone histone stem-loop, and optionally, a poly(A) sequence orpolyadenylation signal. The poly(A) sequence or polyadenylation signalgenerally should enhance the expression level of the encoded protein.The encoded protein, in some embodiments, is not a histone protein, areporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), ora marker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, the RNA vaccine does not comprise a histonedownstream element (HDE). “Histone downstream element” (HDE) includes apurine-rich polynucleotide stretch of approximately 15 to 20 nucleotides3′ of naturally occurring stem-loops, representing the binding site forthe U7 snRNA, which is involved in processing of histone pre-mRNA intomature histone mRNA. Ideally, the inventive nucleic acid does notinclude an intron.

In some embodiments, the RNA vaccine may or may not contain a enhancerand/or promoter sequence, which may be modified or unmodified or whichmay be activated or inactivated. In some embodiments, the histonestem-loop is generally derived from histone genes, and includes anintramolecular base pairing of two neighbored partially or entirelyreverse complementary sequences separated by a spacer, consisting of ashort sequence, which forms the loop of the structure. The unpaired loopregion is typically unable to base pair with either of the stem loopelements. It occurs more often in RNA, as is a key component of many RNAsecondary structures, but may be present in single-stranded DNA as well.Stability of the stem-loop structure generally depends on the length,number of mismatches or bulges, and base composition of the pairedregion. In some embodiments, wobble base pairing (non-Watson-Crick basepairing) may result. In some embodiments, the at least one histonestem-loop sequence comprises a length of 15 to 45 nucleotides.

In other embodiments the RNA vaccine may have one or more AU-richsequences removed. These sequences, sometimes referred to as AURES aredestabilizing sequences found in the 3′UTR. The AURES may be removedfrom the RNA vaccines. Alternatively the AURES may remain in the RNAvaccine.

Nanoparticle Formulations

In some embodiments, HCMV RNA vaccines are formulated in a nanoparticle.In some embodiments, HCMV RNA vaccines are formulated in a lipidnanoparticle. In some embodiments, HCMV RNA vaccines are formulated in alipid-polycation complex, referred to as a cationic lipid nanoparticle.The formation of the lipid nanoparticle may be accomplished by methodsknown in the art and/or as described in U.S. Pub. No. 20120178702,herein incorporated by reference in its entirety. As a non-limitingexample, the polycation may include a cationic peptide or a polypeptidesuch as, but not limited to, polylysine, polyornithine and/orpolyarginine and the cationic peptides described in International Pub.No. WO2012013326 or US Patent Pub. No. US20130142818; each of which isherein incorporated by reference in its entirety. In some embodiments,HCMV RNA vaccines are formulated in a lipid nanoparticle that includes anon-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

A lipid nanoparticle formulation may be influenced by, but not limitedto, the selection of the cationic lipid component, the degree ofcationic lipid saturation, the nature of the PEGylation, ratio of allcomponents and biophysical parameters such as size. In one example bySemple et al. (Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the lipid nanoparticle formulation iscomposed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine,34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid can more effectively deliver siRNA tovarious antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety).

In some embodiments, lipid nanoparticle formulations may comprise 35 to45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipidand/or 55% to 65% cationic lipid. In some embodiments, the ratio oflipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1,10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticleformulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the lipid nanoparticleformulations. As a non-limiting example, lipid nanoparticle formulationsmay contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5%to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, a HCMV RNA vaccine formulation is a nanoparticlethat comprises at least one lipid. The lipid may be selected from, butis not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA,DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and aminoalcohol lipids. In some embodiments, the lipid may be a cationic lipidsuch as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA,DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationiclipid may be the lipids described in and/or made by the methodsdescribed in US Patent Publication No. US20130150625, hereinincorporated by reference in its entirety. As a non-limiting example,the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, a lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, a lipid nanoparticle formulation includes 25% to75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

In some embodiments, a lipid nanoparticle formulation includes 0.5% to15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include,without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments,the formulation includes 5% to 50% on a molar basis of the sterol (e.g.,15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. Anon-limiting example of a sterol is cholesterol. In some embodiments, alipid nanoparticle formulation includes 0.5% to 20% on a molar basis ofthe PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%,1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprisesa PEG molecule of an average molecular weight of less than 2,000, forexample around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limitingexamples of PEG-modified lipids include PEG-distearoyl glycerol(PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA(further discussed in Reyes et al. J. Controlled Release, 107, 276-287(2005) the contents of which are herein incorporated by reference in itsentirety).

In some embodiments, lipid nanoparticle formulations include 25-75% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis. In some embodiments, lipidnanoparticle formulations include 35-65% of a cationic lipid selectedfrom 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 45-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 60% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of theneutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modifiedlipid, and 0.5% of the targeting lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 40% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of theneutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.2% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of theneutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the contents of which are herein incorporated by reference inits entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5%of the PEG or PEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in molar ratios of 20-70% cationiclipid:5-45% neutral lipid:20-55% cholesterol: 0.5-15% PEG-modifiedlipid. In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in a molar ratio of 20-60% cationiclipid:5-25% neutral lipid:25-55% cholesterol: 0.5-15% PEG-modifiedlipid.

In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationiclipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g.,PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid,e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Non-limiting examples of lipid nanoparticle compositions and methods ofmaking them are described, for example, in Semple et al. (2010) Nat.Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed.,51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578(the contents of each of which are incorporated herein by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations may comprise acationic lipid, a PEG lipid and a structural lipid and optionallycomprise a non-cationic lipid. As a non-limiting example, a lipidnanoparticle may comprise 40-60% of cationic lipid, 5-15% of anon-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structurallipid. As another non-limiting example, the lipid nanoparticle maycomprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and38.5% structural lipid. As yet another non-limiting example, a lipidnanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid,2.5% PEG lipid and 32.5% structural lipid. In some embodiments, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2%of a PEG lipid and 30-50% of a structural lipid. As another non-limitingexample, the lipid nanoparticle may comprise 50% cationic lipid, 10%non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle may comprise 55%cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-KC2-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of thestructural lipid cholesterol. As yet another non-limiting example, thelipid nanoparticle comprise 55% of the cationic lipid L319, 10% of thenon-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of thestructural lipid cholesterol.

In some embodiments, a nanoparticle comprises compounds of Formula (I):

or a salt or isomer thereof, wherein:R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R,—S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl andC₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which R₁ is selected from the group consisting of C₅₋₃₀ alkyl,C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)O R, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; M and M′ are independently selected from—C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—,—SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and aheteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl; each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR,—N(R)R₈,—O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂,—N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉)N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycleand (i) R₄ is —(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is—(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, thenQ is either a 5- to 14-membered heteroaryl or 8- to 14-memberedheterocycloalkyl;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle, —(CH₂)Q,—(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q isselected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroaryl havingone or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR,and —C(═NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4,and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂,where Q is —N(R)₂, and n is selected from 1, 2, 3, 4, and 5;each R⁵ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q isOH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (lib), (IIc), or (lie):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (lib), (IIc), or (lie):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In further embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

andsalts and isomers thereof.In some embodiments, a nanoparticle comprises the following compound:

or salts and isomers thereof.

In some embodiments, the disclosure features a nanoparticle compositionincluding a lipid component comprising a compound as described herein(e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe)).

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in a vaccinecomposition may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the composition is to be administered. For example, thecomposition may comprise between 0.1% and 99% (w/w) of the activeingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, atleast 80% (w/w) active ingredient.

In some embodiments, the RNA vaccine composition may comprise thepolynucleotide described herein, formulated in a lipid nanoparticlecomprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodiumcitrate, sucrose and water for injection. As a non-limiting example, thecomposition comprises: 2.0 mg/mL of drug substance (e.g.,polynucleotides encoding H10N8 influenza virus), 21.8 mg/mL of MC3, 10.1mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16mg/mL of trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water forinjection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

Flagellin is an approximately 500 amino acid monomeric protein thatpolymerizes to form the flagella associated with bacterial motion.Flagellin is expressed by a variety of flagellated bacteria (Salmonellatyphimurium for example) as well as non-flagellated bacteria (such asEscherichia coli). Sensing of flagellin by cells of the innate immunesystem (dendritic cells, macrophages, etc.) is mediated by the Toll-likereceptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf andNaip5. TLRs and NLRs have been identified as playing a role in theactivation of innate immune response and adaptive immune response. Assuch, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellinpolypeptides are publicly available in the NCBI GenBank database. Theflagellin sequences from S. Typhimurium, H. Pylori, V. Cholera, S.marcesens, S. flexneri, T. pallidum, L. pneumophila, B. burgdorferei, C.difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.Mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli,among others are known.

A flagellin polypeptide, as used herein, refers to a full lengthflagellin protein, immunogenic fragments thereof, and peptides having atleast 50% sequence identity to a flagellin protein or immunogenicfragments thereof. Exemplary flagellin proteins include flagellin fromSalmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium(A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonellacholeraesuis (Q6V2X8). In some embodiments, the flagellin polypeptidehas at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to a flagellin protein or immunogenic fragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenicfragment. An immunogenic fragment is a portion of a flagellin proteinthat provokes an immune response. In some embodiments, the immuneresponse is a TLR5 immune response. An example of an immunogenicfragment is a flagellin protein in which all or a portion of a hingeregion has been deleted or replaced with other amino acids. For example,an antigenic polypeptide may be inserted in the hinge region. Hingeregions are the hypervariable regions of a flagellin. Hinge regions of aflagellin are also referred to as “D3 domain or region,” “propellerdomain or region,” “hypervariable domain or region” and “variable domainor region.” “At least a portion of a hinge region,” as used herein,refers to any part of the hinge region of the flagellin, or the entiretyof the hinge region. In other embodiments an immunogenic fragment offlagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment offlagellin.

The flagellin monomer is formed by domains D0 through D3. D0 and D1,which form the stem, are composed of tandem long alpha helices and arehighly conserved among different bacteria. The D1 domain includesseveral stretches of amino acids that are useful for TLR5 activation.The entire D1 domain or one or more of the active regions within thedomain are immunogenic fragments of flagellin. Examples of immunogenicregions within the D1 domain include residues 88-114 and residues411-431 in Salmonella typhimurium FliC flagellin. Within the 13 aminoacids in the 88-100 region, at least 6 substitutions are permittedbetween Salmonella flagellin and other flagellins that still preserveTLR5 activation. Thus, immunogenic fragments of flagellin includeflagellin like sequences that activate TLR5 and contain a 13 amino acidmotif that is 53% or more identical to the Salmonella sequence in 88-100of FliC (LQRVRELAVQSAN; SEQ ID NO: 428).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA thatencodes a fusion protein of flagellin and one or more antigenicpolypeptides. A “fusion protein” as used herein, refers to a linking oftwo components of the construct. In some embodiments, a carboxy-terminusof the antigenic polypeptide is fused or linked to an amino terminus ofthe flagellin polypeptide. In other embodiments, an amino-terminus ofthe antigenic polypeptide is fused or linked to a carboxy-terminus ofthe flagellin polypeptide. The fusion protein may include, for example,one, two, three, four, five, six or more flagellin polypeptides linkedto one, two, three, four, five, six or more antigenic polypeptides. Whentwo or more flagellin polypeptides and/or two or more antigenicpolypeptides are linked such a construct may be referred to as a“multimer.”

Each of the components of a fusion protein may be directly linked to oneanother or they may be connected through a linker. For instance, thelinker may be an amino acid linker. The amino acid linker encoded for bythe RNA (e.g., mRNA) vaccine to link the components of the fusionprotein may include, for instance, at least one member selected from thegroup consisting of a lysine residue, a glutamic acid residue, a serineresidue and an arginine residue. In some embodiments the linker is 1-30,1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

In other embodiments the RNA (e.g., mRNA) vaccine includes at least twoseparate RNA polynucleotides, one encoding one or more antigenicpolypeptides and the other encoding the flagellin polypeptide. The atleast two RNA polynucleotides may be co-formulated in a carrier such asa lipid nanoparticle.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The RNA vaccines of the invention can be formulated using one or moreliposomes, lipoplexes, or lipid nanoparticles. In some embodiments, theRNA vaccine comprises one or more RNA polynucleotides comprising one ormore open reading frames encoding one or more of HCMV antigenicpolypeptides gB, gH, gL, UL128, UL130 and UL131. In some embodiments,all of the RNA polynucleotide components of the vaccine are formulatedin the same liposome, lipoplex or lipid nanoparticle. In otherembodiments, one or more of the RNA polynucleotide components of thevaccine are formulated in different liposomes, lipoplexes or lipidnanoparticles. In other embodiments, each of RNA polynucleotidecomponents of the vaccine is formulated in a different liposome,lipoplex or lipid nanoparticle. In some embodiments, an RNA vaccinecomprises RNA polynucleotides encoding gB, gH, gL, UL128, UL130 andUL131. The RNA polynucleotides encoding gB, gH, gL, UL128, UL130 andUL131 can be formulated in one or more liposomes, lipoplexes, or lipidnanoparticles. In certain embodiments, RNA polynucleotides encoding gB,gH, gL, UL128, UL130 and UL131 are all included in the same liposome,lipoplexe, or lipid nanoparticle.

In some embodiments, pharmaceutical compositions of RNA vaccines includeliposomes. Liposomes are artificially-prepared vesicles which mayprimarily be composed of a lipid bilayer and may be used as a deliveryvehicle for the administration of nutrients and pharmaceuticalformulations. Liposomes can be of different sizes such as, but notlimited to, a multilamellar vesicle (MLV) which may be hundreds ofnanometers in diameter and may contain a series of concentric bilayersseparated by narrow aqueous compartments, a small unicellular vesicle(SUV) which may be smaller than 50 nm in diameter, and a largeunilamellar vesicle (LUV) which may be between 50 and 500 nm indiameter. Liposome design may include, but is not limited to, opsoninsor ligands in order to improve the attachment of liposomes to unhealthytissue or to activate events such as, but not limited to, endocytosis.Liposomes may contain a low or a high pH in order to improve thedelivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

As a non-limiting example, liposomes such as synthetic membrane vesiclesmay be prepared by the methods, apparatus and devices described in USPatent Publication No. US20130177638, US20130177637, US20130177636,US20130177635, US20130177634, US20130177633, US20130183375,US20130183373 and US20130183372, the contents of each of which areherein incorporated by reference in its entirety.

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104; all of which are incorporatedherein in their entireties). The original manufacture method by Wheeleret al. was a detergent dialysis method, which was later improved byJeffs et al. and is referred to as the spontaneous vesicle formationmethod. The liposome formulations are composed of 3 to 4 lipidcomponents in addition to the polynucleotide. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations may contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations may comprise from about about25.0% cholesterol to about 40.0% cholesterol, from about 30.0%cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol toabout 50.0% cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. In a preferred embodiment, formulations may comprise apercentage of cholesterol selected from the group consisting of 28.5%,31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments,formulations may comprise from about 5.0% to about 10.0% DSPC and/orfrom about 7.0% to about 15.0% DSPC.

In some embodiments, pharmaceutical compositions may include liposomeswhich may be formed to deliver polynucleotides which may encode at leastone immunogen (antigen) or any other polypeptide of interest. The RNAvaccine may be encapsulated by the liposome and/or it may be containedin an aqueous core which may then be encapsulated by the liposome (seeInternational Pub. Nos. WO2012031046, WO2012031043, WO2012030901 andWO2012006378 and US Patent Publication No. US20130189351, US20130195969and US20130202684; the contents of each of which are herein incorporatedby reference in their entirety).

In another embodiment, liposomes may be formulated for targeteddelivery. As a non-limiting example, the liposome may be formulated fortargeted delivery to the liver. The liposome used for targeted deliverymay include, but is not limited to, the liposomes described in andmethods of making liposomes described in US Patent Publication No.US20130195967, the contents of which are herein incorporated byreference in its entirety.

In another embodiment, the polynucleotide which may encode an immunogen(antigen) may be formulated in a cationic oil-in-water emulsion wherethe emulsion particle comprises an oil core and a cationic lipid whichcan interact with the polynucleotide anchoring the molecule to theemulsion particle (see International Pub. No. WO2012006380; hereinincorporated by reference in its entirety).

In some embodiments, the RNA vaccines may be formulated in awater-in-oil emulsion comprising a continuous hydrophobic phase in whichthe hydrophilic phase is dispersed. As a non-limiting example, theemulsion may be made by the methods described in InternationalPublication No. WO201087791, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the lipid formulation may include at leastcationic lipid, a lipid which may enhance transfection and a least onelipid which contains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; thecontents of each of which is herein incorporated by reference in theirentirety). In another embodiment, the polynucleotides encoding animmunogen may be formulated in a lipid vesicle which may have crosslinksbetween functionalized lipid bilayers (see U.S. Pub. No. 20120177724,the contents of which is herein incorporated by reference in itsentirety).

In some embodiments, the polylnucleotides may be formulated in a lipsomeas described in International Patent Publication No. WO2013086526, thecontents of which is herein incorporated by reference in its entirety.The RNA vaccines may be encapsulated in a liposome using reverse pHgradients and/or optimized internal buffer compositions as described inInternational Patent Publication No. WO2013086526, the contents of whichis herein incorporated by reference in its entirety.

In some embodiments, the RNA vaccine pharmaceutical compositions may beformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713); herein incorporated by referencein its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics,Israel).

In some embodiments, the cationic lipid may be a low molecular weightcationic lipid such as those described in US Patent Application No.20130090372, the contents of which are herein incorporated by referencein its entirety.

In some embodiments, the RNA vaccines may be formulated in a lipidvesicle which may have crosslinks between functionalized lipid bilayers.

In some embodiments, the RNA vaccines may be formulated in a liposomecomprising a cationic lipid. The liposome may have a molar ratio ofnitrogen atoms in the cationic lipid to the phophates in the RNA (N:Pratio) of between 1:1 and 20:1 as described in International PublicationNo. WO2013006825, herein incorporated by reference in its entirety. Inanother embodiment, the liposome may have a N:P ratio of greater than20:1 or less than 1:1.

In some embodiments, the RNA vaccines may be formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished by methods known in the art and/or as described inU.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012013326 or US Patent Pub. No.US20130142818; each of which is herein incorporated by reference in itsentirety. In another embodiment, the RNA vaccines may be formulated in alipid-polycation complex which may further include a non-cationic lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

In some embodiments, the RNA vaccines may be formulated in anaminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in thepresent invention may be prepared by the methods described in U.S. Pat.No. 8,450,298, herein incorporated by reference in its entirety.

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety). In someembodiments, liposome formulations may comprise from about 35 to about45% cationic lipid, from about 40% to about 50% cationic lipid, fromabout 50% to about 60% cationic lipid and/or from about 55% to about 65%cationic lipid. In some embodiments, the ratio of lipid to mRNA inliposomes may be from about about 5:1 to about 20:1, from about 10:1 toabout 25:1, from about 15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In another embodiment the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, the RNA vaccines may be formulated in a lipidnanoparticle such as those described in International Publication No.WO2012170930, the contents of which is herein incorporated by referencein its entirety.

In some embodiments, the RNA vaccine formulation comprising thepolynucleotide is a nanoparticle which may comprise at least one lipid.The lipid may be selected from, but is not limited to, DLin-DMA,DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA,PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In anotheraspect, the lipid may be a cationic lipid such as, but not limited to,DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and aminoalcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in US PatentPublication No. US20130150625, herein incorporated by reference in itsentirety. As a non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, the formulation includes from about 25% to about75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In some embodiments, the formulation includes from about 0.5% to about15% on a molar basis of the neutral lipid e.g., from about 3 to about12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% ona molar basis. Exemplary neutral lipids include, but are not limited to,DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In some embodiments, the formulation includes from about0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid(e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis. In someembodiments, the PEG or PEG modified lipid comprises a PEG molecule ofan average molecular weight of 2,000 Da. In other embodiments, the PEGor PEG modified lipid comprises a PEG molecule of an average molecularweight of less than 2,000, for example around 1,500 Da, around 1,000 Da,or around 500 Da. Exemplary PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J.Controlled Release, 107, 276-287 (2005) the contents of which are hereinincorporated by reference in its entirety)

In some embodiments, the formulations of the inventions include 25-75%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include 35-65%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include 45-65%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include about60% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include about50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include about50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In some embodiments, the formulations of the inventions include about40% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include about57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the inventions include about57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005), the contents of which are herein incorporated byreference in its entirety), about 7.5% of the neutral lipid, about 31.5%of the sterol, and about 3.5% of the PEG or PEG-modified lipid on amolar basis.

In preferred embodiments, lipid nanoparticle formulation consistsessentially of a lipid mixture in molar ratios of about 20-70% cationiclipid:5-45% neutral lipid:20-55% cholesterol: 0.5-15% PEG-modifiedlipid; more preferably in a molar ratio of about 20-60% cationiclipid:5-25% neutral lipid:25-55% cholesterol: 0.5-15% PEG-modifiedlipid.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary lipid nanoparticle compositions and methods of making same aredescribed, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a PEG lipid and a structural lipidand optionally comprise a non-cationic lipid. As a non-limiting example,the lipid nanoparticle may comprise about 40-60% of cationic lipid,about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about30-50% of a structural lipid. As another non-limiting example, the lipidnanoparticle may comprise about 50% cationic lipid, about 10%non-cationic lipid, about 1.5% PEG lipid and about 38.5% structurallipid. As yet another non-limiting example, the lipid nanoparticle maycomprise about 55% cationic lipid, about 10% non-cationic lipid, about2.5% PEG lipid and about 32.5% structural lipid. In some embodiments,the cationic lipid may be any cationic lipid described herein such as,but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise about 40-60% of cationic lipid, about 5-15% of a non-cationiclipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.As another non-limiting example, the lipid nanoparticle may compriseabout 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEGlipid and about 38.5% structural lipid. As yet another non-limitingexample, the lipid nanoparticle may comprise about 55% cationic lipid,about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise about 50% of the cationic lipid DLin-KC2-DMA,about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipidPEG-DOMG and about 38.5% of the structural lipid cholesterol. As anon-limiting example, the lipid nanoparticle comprise about 50% of thecationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC,about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structurallipid cholesterol. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In some embodiments, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent PublicationNo. US20100036115, US20120202871, US20130064894, US20130129785,US20130150625, US20130178541 and US20130225836; the contents of each ofwhich are herein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid may be selected from, but not limited to,formula A described in International Publication Nos. WO2012040184,WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460,WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US PatentPublication No. US20130178541 and US20130225836; the contents of each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.

WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US PatentPublication No. US20100036115, formula I of US Patent Publication NoUS20130123338; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid may beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-1 6, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety.

In another embodiment, the lipid may be a cationic lipid such as, butnot limited to, Formula (I) of U.S. Patent Application No.US20130064894, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2013086373 and WO2013086354; the contents of each ofwhich are herein incorporated by reference in their entirety.

In another embodiment, the cationic lipid may be a trialkyl cationiclipid. Non-limiting examples of trialkyl cationic lipids and methods ofmaking and using the trialkyl cationic lipids are described inInternational Patent Publication No. WO2013126803, the contents of whichare herein incorporated by reference in its entirety.

In some embodiments, the LNP formulations of the RNA vaccines maycontain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment, theLNP formulations RRNA vaccines may contain PEG-c-DOMG at 1.5% lipidmolar ratio.

In some embodiments, the pharmaceutical compositions of the RNA vaccinesmay include at least one of the PEGylated lipids described inInternational Publication No. WO2012099755, the contents of which isherein incorporated by reference in its entirety.

In some embodiments, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In some embodiments, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral deliveryof self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; hereinincorporated by reference in its entirety).

In some embodiments, the LNP formulation may be formulated by themethods described in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which is herein incorporated byreference in their entirety. As a non-limiting example, the RNA vaccinesdescribed herein may be encapsulated in LNP formulations as described inWO2011127255 and/or WO2008103276; each of which is herein incorporatedby reference in their entirety.

In some embodiments, the RNA vaccines described herein may be formulatedin a nanoparticle to be delivered by a parenteral route as described inU.S. Pub. No.

US20120207845; the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the RNA vaccines may be formulated in a lipidnanoparticle made by the methods described in US Patent Publication NoUS20130156845 or International Publication No WO2013093648 orWO2012024526, each of which is herein incorporated by reference in itsentirety.

The lipid nanoparticles described herein may be made in a sterileenvironment by the system and/or methods described in US PatentPublication No. US20130164400, herein incorporated by reference in itsentirety.

In some embodiments, the LNP formulation may be formulated in ananoparticle such as a nucleic acid-lipid particle described in U.S.Pat. No. 8,492,359, the contents of which are herein incorporated byreference in its entirety. As a non-limiting example, the lipid particlemay comprise one or more active agents or therapeutic agents; one ormore cationic lipids comprising from about 50 mol % to about 85 mol % ofthe total lipid present in the particle; one or more non-cationic lipidscomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and one or more conjugated lipids that inhibitaggregation of particles comprising from about 0.5 mol % to about 2 mol% of the total lipid present in the particle. The nucleic acid in thenanoparticle may be the polynucleotides described herein and/or areknown in the art.

In some embodiments, the LNP formulation may be formulated by themethods described in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which are herein incorporated byreference in their entirety. As a non-limiting example, modified RNAdescribed herein may be encapsulated in LNP formulations as described inWO2011127255 and/or WO2008103276; the contents of each of which areherein incorporated by reference in their entirety.

In some embodiments, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety. In another embodiment, the LNP formulationscomprising a polycationic composition may be used for the delivery ofthe modified RNA described herein in vivo and/or in vitro.

In some embodiments, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety.

In some embodiments, the RNA vaccine pharmaceutical compositions may beformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713); herein incorporated by referencein its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics,Israel).

In some embodiments, the RNA vaccines may be formulated in a lyophilizedgel-phase liposomal composition as described in US Publication No.US2012060293, herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present invention may be made by the methods describedin International Application No. WO2013033438 or US Patent PublicationNo. US20130196948, the contents of each of which are herein incorporatedby reference in its entirety. As a non-limiting example, the phosphateconjugates may include a compound of any one of the formulas describedin International Application No. WO2013033438, herein incorporated byreference in its entirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water soluble conjugate. The polymerconjugate may have a structure as described in U.S. Patent ApplicationNo. 20130059360, the contents of which are herein incorporated byreference in its entirety. In one aspect, polymer conjugates with thepolynucleotides of the present invention may be made using the methodsand/or segmented polymeric reagents described in U.S. Patent ApplicationNo. 20130072709, herein incorporated by reference in its entirety. Inanother aspect, the polymer conjugate may have pendant side groupscomprising ring moieties such as, but not limited to, the polymerconjugates described in US Patent Publication No. US20130196948, thecontents of which is herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present invention in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate may be a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al (Science 2013 339, 971-975),herein incorporated by reference in its entirety). As shown by Rodriguezet al. the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles. In anotheraspect, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In some embodiments, the RNA vaccines of the present invention areformulated in nanoparticles which comprise a conjugate to enhance thedelivery of the nanoparticles of the present invention in a subject. Theconjugate may be the CD47 membrane or the conjugate may be derived fromthe CD47 membrane protein, such as the “self” peptide describedpreviously. In another aspect the nanoparticle may comprise PEG and aconjugate of CD47 or a derivative thereof. In yet another aspect, thenanoparticle may comprise both the “self” peptide described above andthe membrane protein CD47.

In another aspect, a “self” peptide and/or CD47 protein may beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the RNA vaccines of the present invention.

In another embodiment, RNA vaccine pharmaceutical compositionscomprising the polynucleotides of the present invention and a conjugatewhich may have a degradable linkage. Non-limiting examples of conjugatesinclude an aromatic moiety comprising an ionizable hydrogen atom, aspacer moiety, and a water-soluble polymer. As a non-limiting example,pharmaceutical compositions comprising a conjugate with a degradablelinkage and methods for delivering such pharmaceutical compositions aredescribed in US Patent Publication No. US20130184443, the contents ofwhich are herein incorporated by reference in its entirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a RNA vaccine. As a non-limitingexample, the carbohydrate carrier may include, but is not limited to, ananhydride-modified phytoglycogen or glycogen-type material, phtoglycogenoctenyl succinate, phytoglycogen beta-dextrin, anhydride-modifiedphytoglycogen beta-dextrin. (See e.g., International Publication No.WO2012109121; the contents of which are herein incorporated by referencein its entirety).

Nanoparticle formulations of the present invention may be coated with asurfactant or polymer in order to improve the delivery of the particle.In some embodiments, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,RNA vaccines within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in US Patent Publication No.US20130183244, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the lipid nanoparticles of the present inventionmay be hydrophilic polymer particles. Non-limiting examples ofhydrophilic polymer particles and methods of making hydrophilic polymerparticles are described in US Patent Publication No. US20130210991, thecontents of which are herein incorporated by reference in its entirety.

In another embodiment, the lipid nanoparticles of the present inventionmay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In some embodiments, the internal ester linkage may be located on eitherside of the saturated carbon.

In some embodiments, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a polynucleotidedescribed herein. In some embodiments, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670 or International PatentPublication No. WO2013110028, the contents of each of which are hereinincorporated by reference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Patent PublicationNo. WO2013116804, the contents of which are herein incorporated byreference in its entirety. The polymeric material may additionally beirradiated. As a non-limiting example, the polymeric material may begamma irradiated (See e.g., International App. No. WO201282165, hereinincorporated by reference in its entirety). Non-limiting examples ofspecific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., USPublication 20120121718 and US Publication 20100003337 and U.S. Pat. No.8,263,665; each of which is herein incorporated by reference in theirentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; thecontents of which are herein incorporated by reference in its entirety).A non-limiting scalable method to produce nanoparticles which canpenetrate human mucus is described by Xu et al. (See e.g., J ControlRelease 2013, 170(2):279-86; the contents of which are hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecylammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase. The surface altering agent may be embedded orenmeshed in the particle's surface or disposed (e.g., by coating,adsorption, covalent linkage, or other process) on the surface of thelipid nanoparticle. (see e.g., US Publication 20100215580 and USPublication 20080166414 and US20130164343; the contents of each of whichis herein incorporated by reference in their entirety).

In some embodiments, the mucus penetrating lipid nanoparticles maycomprise at least one polynucleotide described herein. Thepolynucleotide may be encapsulated in the lipid nanoparticle and/ordisposed on the surface of the particle. The polynucleotide may becovalently coupled to the lipid nanoparticle. Formulations of mucuspenetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In another embodiment, the mucus penetrating lipid nanoparticles may bea hypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation may be hypotonice for the epithelium to whichit is being delivered. Non-limiting examples of hypotonic formulationsmay be found in International Patent Publication No. WO2013110028, thecontents of which are herein incorporated by reference in its entirety.

In some embodiments, in order to enhance the delivery through themucosal barrier the RNA vaccine formulation may comprise or be ahypotonic solution. Hypotonic solutions were found to increase the rateat which mucoinert particles such as, but not limited to,mucus-penetrating particles, were able to reach the vaginal epithelialsurface (See e.g., Ensign et al. Biomaterials 2013 34(28):6922-9; thecontents of which is herein incorporated by reference in its entirety).

In some embodiments, the RNA vaccine is formulated as a lipoplex, suchas, without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids acids (Aleku et al. Cancer Res. 200868:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78;Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. JImmunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100;deFougerolles Hum Gene Ther. 2008 19:125-132; all of which areincorporated herein by reference in its entirety).

In some embodiments such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In some embodiments, the RNA vaccine is formulated as a solid lipidnanoparticle. A solid lipid nanoparticle (SLN) may be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and may be stabilizedwith surfactants and/or emulsifiers. In a further embodiment, the lipidnanoparticle may be a self-assembly lipid-polymer nanoparticle (seeZhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of whichare herein incorporated by reference in its entirety). As a non-limitingexample, the SLN may be the SLN described in International PatentPublication No. WO2013105101, the contents of which are hereinincorporated by reference in its entirety. As another non-limitingexample, the SLN may be made by the methods or processes described inInternational Patent Publication No. WO2013105101, the contents of whichare herein incorporated by reference in its entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides directed protein production as theseformulations may be able to increase cell transfection by the RNAvaccine; and/or increase the translation of encoded protein. One suchexample involves the use of lipid encapsulation to enable the effectivesystemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 200715:713-720; herein incorporated by reference in its entirety). Theliposomes, lipoplexes, or lipid nanoparticles may also be used toincrease the stability of the polynucleotide.

In some embodiments, the RNA vaccines of the present invention can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In some embodiments, the RRNAvaccines may be encapsulated into a delivery agent described hereinand/or known in the art for controlled release and/or targeted delivery.As used herein, the term “encapsulate” means to enclose, surround orencase. As it relates to the formulation of the compounds of theinvention, encapsulation may be substantial, complete or partial. Theterm “substantially encapsulated” means that at least greater than 50,60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than99.999% of the pharmaceutical composition or compound of the inventionmay be enclosed, surrounded or encased within the delivery agent.“Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 orless of the pharmaceutical composition or compound of the invention maybe enclosed, surrounded or encased within the delivery agent.Advantageously, encapsulation may be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of theinvention using fluorescence and/or electron micrograph. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,99.9, 99.99 or greater than 99.99% of the pharmaceutical composition orcompound of the invention are encapsulated in the delivery agent.

In some embodiments, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; the contents ofeach of which is herein incorporated by reference in its entirety).

In another embodiment, the RNA vaccines may be encapsulated into a lipidnanoparticle or a rapidly eliminated lipid nanoparticle and the lipidnanoparticles or a rapidly eliminated lipid nanoparticle may then beencapsulated into a polymer, hydrogel and/or surgical sealant describedherein and/or known in the art. As a non-limiting example, the polymer,hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc),poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX®(Halozyme Therapeutics, San Diego Calif.), surgical sealants such asfibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (BaxterInternational, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL®(Baxter International, Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In some embodiments, the the RNA vaccine formulation for controlledrelease and/or targeted delivery may also include at least onecontrolled release coating. Controlled release coatings include, but arenot limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® andcellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In some embodiments, the RNA vaccine controlled release and/or targeteddelivery formulation may comprise at least one degradable polyesterwhich may contain polycationic side chains. Degradeable polyestersinclude, but are not limited to, poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), andcombinations thereof. In another embodiment, the degradable polyestersmay include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the RNA vaccine controlled release and/or targeteddelivery formulation comprising at least one polynucleotide may compriseat least one PEG and/or PEG related polymer derivatives as described inU.S. Pat. No. 8,404,222, herein incorporated by reference in itsentirety.

In another embodiment, the RNA vaccine controlled release deliveryformulation comprising at least one polynucleotide may be the controlledrelease polymer system described in US20130130348, herein incorporatedby reference in its entirety.

In some embodiments, the the RNA vaccines of the present invention maybe encapsulated in a therapeutic nanoparticle, referred to herein as“therapeutic nanoparticle RRNA vaccines.” Therapeutic nanoparticles maybe formulated by methods described herein and known in the art such as,but not limited to, International Pub Nos. WO2010005740, WO2010030763,WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491,US20100104645, US20100087337, US20100068285, US20110274759,US20100068286, US20120288541, US20130123351 and US20130230567 and U.S.Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents ofeach of which are herein incorporated by reference in their entirety. Inanother embodiment, therapeutic polymer nanoparticles may be identifiedby the methods described in US Pub No. US20120140790, the contents ofwhich is herein incorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle RNA vaccine may beformulated for sustained release. As used herein, “sustained release”refers to a pharmaceutical composition or compound that conforms to arelease rate over a specific period of time. The period of time mayinclude, but is not limited to, hours, days, weeks, months and years. Asa non-limiting example, the sustained release nanoparticle may comprisea polymer and a therapeutic agent such as, but not limited to, the thepolynucleotides of the present invention (see International Pub No.2010075072 and US Pub No. US20100216804, US20110217377 andUS20120201859, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the sustained releaseformulation may comprise agents which permit persistent bioavailabilitysuch as, but not limited to, crystals, macromolecular gels and/orparticulate suspensions (see US Patent Publication No US20130150295, thecontents of which is herein incorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle RNA vaccines may beformulated to be target specific. As a non-limiting example, thethereapeutic nanoparticles may include a corticosteroid (seeInternational Pub. No. WO2011084518; herein incorporated by reference inits entirety). As a non-limiting example, the therapeutic nanoparticlesmay be formulated in nanoparticles described in International Pub No.WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No.US20100069426, US20120004293 and US20100104655, each of which is hereinincorporated by reference in their entirety.

In some embodiments, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In some embodiments, the therapeutic nanoparticle comprises a diblockcopolymer. In some embodiments, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In anotherembodiment, the diblock copolymer may comprise the diblock copolymersdescribed in European Patent Publication No. the contents of which areherein incorporated by reference in its entirety. In yet anotherembodiment, the diblock copolymer may be a high-X diblock copolymer suchas those described in International Patent Publication No. WO2013120052,the contents of which are herein incorporated by reference in itsentirety.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 andInternational Publication No. WO2012166923, the contents of each ofwhich are herein incorporated by reference in its entirety). In yetanother non-limiting example, the therapeutic nanoparticle is a stealthnanoparticle or a target-specific stealth nanoparticle as described inUS Patent Publication No. US20130172406, the contents of which areherein incorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and US Patent Pub. No. US20130195987; the contents of each of which areherein incorporated by reference in its entirety).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) was used as a TGF-betal gene delivery vehicle in Lee etal. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000;as a controlled gene delivery system in Li et al. Controlled GeneDelivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253; each of which is herein incorporated by reference in itsentirety). The RNA vaccines of the present invention may be formulatedin lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and US Patent Pub. No. US20130195987; the contents of each of which areherein incorporated by reference in its entirety).

In some embodiments, the block copolymers described herein may beincluded in a polyion complex comprising a non-polymeric micelle and theblock copolymer. (See e.g., U.S. Pub. No. 20120076836; hereinincorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In some embodiments, the therapeutic nanoparticles may comprise at leastone poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer may have a structure such as those described inInternational Application No. WO2013032829 or US Patent Publication NoUS20130121954, the contents of which are herein incorporated byreference in its entirety. In one aspect, the poly(vinyl ester) polymersmay be conjugated to the polynucleotides described herein. In anotheraspect, the poly(vinyl ester) polymer which may be used in the presentinvention may be those described in, herein incorporated by reference inits entirety.

In some embodiments, the therapeutic nanoparticle may comprise at leastone diblock copolymer. The diblock copolymer may be, but it not limitedto, a poly(lactic) acid-poly(ethylene)glycol copolymer (see e.g.,International Patent Publication No. WO2013044219; herein incorporatedby reference in its entirety). As a non-limiting example, thetherapeutic nanoparticle may be used to treat cancer (see Internationalpublication No. WO2013044219; herein incorporated by reference in itsentirety).

In some embodiments, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In some embodiments, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference inits entirety) and combinations thereof.

In another embodiment, the nanoparticles described herein may comprisean amine cationic lipid such as those described in International PatentApplication No. WO2013059496, the contents of which are hereinincorporated by reference in its entirety. In one aspect the cationiclipids may have an amino-amine or an amino-amide moiety.

In some embodiments, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand. The targeting ligand maybe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; hereinincorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle may be formulated inan aqueous solution which may be used to target cancer (seeInternational Pub No. WO2011084513 and US Pub No. US20110294717, each ofwhich is herein incorporated by reference in their entirety).

In some embodiments, the therapeutic nanoparticle RNA vaccines, e.g.,therapeutic nanoparticles comprising at least one RNA vaccine may beformulated using the methods described by Podobinski et al in U.S. Pat.No. 8,404,799, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the RNA vaccines may be encapsulated in, linked toand/or associated with synthetic nanocarriers. Synthetic nanocarriersinclude, but are not limited to, those described in International Pub.Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252,WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282,WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 andWO2013019669, and US Pub. Nos. US20110262491, US20100104645,US20100087337 and US20120244222, each of which is herein incorporated byreference in their entirety. The synthetic nanocarriers may beformulated using methods known in the art and/or described herein. As anon-limiting example, the synthetic nanocarriers may be formulated bythe methods described in International Pub Nos. WO2010005740,WO2010030763 and WO201213501 and US Pub. Nos. US20110262491,US20100104645, US20100087337 and US2012024422, each of which is hereinincorporated by reference in their entirety. In another embodiment, thesynthetic nanocarrier formulations may be lyophilized by methodsdescribed in International Pub. No. WO2011072218 and U.S. Pat. No.8,211,473; the content of each of which is herein incorporated byreference in their entirety. In yet another embodiment, formulations ofthe present invention, including, but not limited to, syntheticnanocarriers, may be lyophilized or reconstituted by the methodsdescribed in US Patent Publication No. US20130230568, the contents ofwhich are herein incorporated by reference in its entirety.

In some embodiments, the synthetic nanocarriers may contain reactivegroups to release the polynucleotides described herein (seeInternational Pub. No. WO20120952552 and US Pub No. US20120171229, eachof which is herein incorporated by reference in their entirety).

In some embodiments, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In some embodiments, the synthetic nanocarriers may be formulated fortargeted release. In some embodiments, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle may be formulated to release the RNA vaccines after 24hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, eachof which is herein incorporated by reference in their entireties).

In some embodiments, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release may be formulated by methods known in the art,described herein and/or as described in International Pub No.WO2010138192 and US Pub No. 20100303850, each of which is hereinincorporated by reference in their entirety.

In some embodiments, the RNA vaccine may be formulated for controlledand/or sustained release wherein the formulation comprises at least onepolymer that is a crystalline side chain (CYSC) polymer. CYSC polymersare described in U.S. Pat. No. 8,399,007, herein incorporated byreference in its entirety.

In some embodiments, the synthetic nanocarrier may be formulated for useas a vaccine. In some embodiments, the synthetic nanocarrier mayencapsulate at least one polynucleotide which encode at least oneantigen. As a non-limiting example, the synthetic nanocarrier mayinclude at least one antigen and an excipient for a vaccine dosage form(see International Pub No. WO2011150264 and US Pub No. US20110293723,each of which is herein incorporated by reference in their entirety). Asanother non-limiting example, a vaccine dosage form may include at leasttwo synthetic nanocarriers with the same or different antigens and anexcipient (see International Pub No. WO2011150249 and US Pub No.US20110293701, each of which is herein incorporated by reference intheir entirety). The vaccine dosage form may be selected by methodsdescribed herein, known in the art and/or described in International PubNo. WO2011150258 and US Pub No. US20120027806, each of which is hereinincorporated by reference in their entirety).

In some embodiments, the synthetic nanocarrier may comprise at least onepolynucleotide which encodes at least one adjuvant. As non-limitingexample, the adjuvant may comprise dimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium (Seee.g, U.S. Pat. No. 8,241,610; herein incorporated by reference in itsentirety). In another embodiment, the synthetic nanocarrier may compriseat least one polynucleotide and an adjuvant. As a non-limiting example,the synthetic nanocarrier comprising and adjuvant may be formulated bythe methods described in International Pub No. WO2011150240 and US PubNo. US20110293700, each of which is herein incorporated by reference inits entirety.

In some embodiments, the synthetic nanocarrier may encapsulate at leastone polynucleotide which encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier may include,but is not limited to, the nanocarriers described in International PubNo. WO2012024621, WO201202629, WO2012024632 and US Pub No.US20120064110, US20120058153 and US20120058154, each of which is hereinincorporated by reference in their entirety.

In some embodiments, the synthetic nanocarrier may be coupled to apolynucleotide which may be able to trigger a humoral and/or cytotoxic Tlymphocyte (CTL) response (See e.g., International Publication No.WO2013019669, herein incorporated by reference in its entirety).

In some embodiments, the RNA vaccine may be encapsulated in, linked toand/or associated with zwitterionic lipids. Non-limiting examples ofzwitterionic lipids and methods of using zwitterionic lipids aredescribed in US Patent Publication No. US20130216607, the contents ofwhich are herein incorporated by reference in its entirety. In oneaspect, the zwitterionic lipids may be used in the liposomes and lipidnanoparticles described herein.

In some embodiments, the RNA vaccine may be formulated in colloidnanocarriers as described in US Patent Publication No. US20130197100,the contents of which are herein incorporated by reference in itsentirety.

In some embodiments, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832 expresslyincorporated herein by reference in its entirety). Activity and/orsafety (as measured by examining one or more of ALT/AST, white bloodcell count and cytokine induction) of LNP administration may be improvedby incorporation of such lipids. LNPs comprising KL52 may beadministered intravenously and/or in one or more doses. In someembodiments, administration of LNPs comprising KL52 results in equal orimproved mRNA and/or protein expression as compared to LNPs comprisingMC3.

In some embodiments, RNA vaccine may be delivered using smaller LNPs.Such particles may comprise a diameter from below 0.1 um up to 100 nmsuch as, but not limited to, less than 0.1 um, less than 1.0 um, lessthan 5 um, less than 10 um, less than 15 um, less than 20 um, less than25 um, less than 30 um, less than 35 um, less than 40 um, less than 50um, less than 55 um, less than 60 um, less than 65 um, less than 70 um,less than 75 um, less than 80 um, less than 85 um, less than 90 um, lessthan 95 um, less than 100 um, less than 125 um, less than 150 um, lessthan 175 um, less than 200 um, less than 225 um, less than 250 um, lessthan 275 um, less than 300 um, less than 325 um, less than 350 um, lessthan 375 um, less than 400 um, less than 425 um, less than 450 um, lessthan 475 um, less than 500 um, less than 525 um, less than 550 um, lessthan 575 um, less than 600 um, less than 625 um, less than 650 um, lessthan 675 um, less than 700 um, less than 725 um, less than 750 um, lessthan 775 um, less than 800 um, less than 825 um, less than 850 um, lessthan 875 um, less than 900 um, less than 925 um, less than 950 um, andless than 975 um.

In another embodiment, RNA vaccines may be delivered using smaller LNPswhich may comprise a diameter from about 1 nm to about 100 nm, fromabout 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm toabout 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, fromabout 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm toabout 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, fromabout 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50nm, from about 30 to about 50 nm, from about 40 to about 50 nm, fromabout 20 to about 60 nm, from about 30 to about 60 nm, from about 40 toabout 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm,from about 40 to about 70 nm, from about 50 to about 70 nm, from about60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, fromabout 60 to about 80 nm, from about 20 to about 90 nm, from about 30 toabout 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm,from about 60 to about 90 nm and/or from about 70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Exemplary microfluidic mixers may include, but arenot limited to a slit interdigitial micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51; each of which is herein incorporated by reference inits entirety). In some embodiments, methods of LNP generation comprisingSHM, further comprise the mixing of at least two input streams whereinmixing occurs by microstructure-induced chaotic advection (MICA).According to this method, fluid streams flow through channels present ina herringbone pattern causing rotational flow and folding the fluidsaround each other. This method may also comprise a surface for fluidmixing wherein the surface changes orientations during fluid cycling.Methods of generating LNPs using SHM include those disclosed in U.S.Application Publication Nos. 2004/0262223 and 2012/0276209, each ofwhich is expressly incorporated herein by reference in their entirety.

In some embodiments, the RNA vaccine of the present invention may beformulated in lipid nanoparticles created using a micromixer such as,but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2)or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar(CPMM) or Impinging-jet (IJMM) from the Institut fur Mikrotechnik MainzGmbH, Mainz Germany).

In some embodiments, the RNA vaccines of the present invention may beformulated in lipid nanoparticles created using microfluidic technology(see Whitesides, George M. The Origins and the Future of Microfluidics.Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; each of which is hereinincorporated by reference in its entirety). As a non-limiting example,controlled microfluidic formulation includes a passive method for mixingstreams of steady pressure-driven flows in micro channels at a lowReynolds number (See e.g., Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; which is herein incorporatedby reference in its entirety).

In some embodiments, the RNA vaccines of the present invention may beformulated in lipid nanoparticles created using a micromixer chip suchas, but not limited to, those from Harvard Apparatus (Holliston, Mass.)or Dolomite Microfluidics (Royston, UK). A micromixer chip can be usedfor rapid mixing of two or more fluid streams with a split and recombinemechanism.

In some embodiments, the RNA vaccines of the invention may be formulatedfor delivery using the drug encapsulating microspheres described inInternational Patent Publication No. WO2013063468 or U.S. Pat. No.8,440,614, each of which is herein incorporated by reference in itsentirety. The microspheres may comprise a compound of the formula (I),(II), (III), (IV), (V) or (VI) as described in International PatentPublication No. WO2013063468, the contents of which are hereinincorporated by reference in its entirety. In another aspect, the aminoacid, peptide, polypeptide, lipids (APPL) are useful in delivering theRNA vaccines of the invention to cells (see International PatentPublication No. WO2013063468, the contents of which is hereinincorporated by reference in its entirety).

In some embodiments, the RNA vaccines of the invention may be formulatedin lipid nanoparticles having a diameter from about 10 to about 100 nmsuch as, but not limited to, about 10 to about 20 nm, about 10 to about30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 toabout 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm,about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 toabout 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm,about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 toabout 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100nm.

In some embodiments, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In some embodiments, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In one aspect, the lipid nanoparticle may be a limit size lipidnanoparticle described in International Patent Publication No.WO2013059922, the contents of which are herein incorporated by referencein its entirety. The limit size lipid nanoparticle may comprise a lipidbilayer surrounding an aqueous core or a hydrophobic core; where thelipid bilayer may comprise a phospholipid such as, but not limited to,diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide,a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, aC8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect the limit size lipidnanoparticle may comprise a polyethylene glycol-lipid such as, but notlimited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In some embodiments, the RNA vaccines may be delivered, localized and/orconcentrated in a specific location using the delivery methods describedin International Patent Publication No. WO2013063530, the contents ofwhich are herein incorporated by reference in its entirety. As anon-limiting example, a subject may be administered an empty polymericparticle prior to, simultaneously with or after delivering the RNAvaccines to the subject. The empty polymeric particle undergoes a changein volume once in contact with the subject and becomes lodged, embedded,immobilized or entrapped at a specific location in the subject.

In some embodiments, the RNA vaccines may be formulated in an activesubstance release system (See e.g., US Patent Publication No.US20130102545, the contents of which is herein incorporated by referencein its entirety). The active substance release system may comprise 1) atleast one nanoparticle bonded to an oligonucleotide inhibitor strandwhich is hybridized with a catalytically active nucleic acid and 2) acompound bonded to at least one substrate molecule bonded to atherapeutically active substance (e.g., polynucleotides describedherein), where the therapeutically active substance is released by thecleavage of the substrate molecule by the catalytically active nucleicacid.

In some embodiments, the RNA vaccines may be formulated in ananoparticle comprising an inner core comprising a non-cellular materialand an outer surface comprising a cellular membrane. The cellularmembrane may be derived from a cell or a membrane derived from a virus.As a non-limiting example, the nanoparticle may be made by the methodsdescribed in International Patent Publication No. WO2013052167, hereinincorporated by reference in its entirety. As another non-limitingexample, the nanoparticle described in International Patent PublicationNo. WO2013052167, herein incorporated by reference in its entirety, maybe used to deliver the RNA vaccines described herein.

In some embodiments, the RNA vaccines may be formulated in porousnanoparticle-supported lipid bilayers (protocells). Protocells aredescribed in International Patent Publication No. WO2013056132, thecontents of which are herein incorporated by reference in its entirety.

In some embodiments, the RNA vaccines described herein may be formulatedin polymeric nanoparticles as described in or made by the methodsdescribed in U.S. Pat. Nos. 8,420,123 and 8,518,963 and European PatentNo. EP2073848B1, the contents of each of which are herein incorporatedby reference in their entirety. As a non-limiting example, the polymericnanoparticle may have a high glass transition temperature such as thenanoparticles described in or nanoparticles made by the methodsdescribed in U.S. Pat. No. 8,518,963, the contents of which are hereinincorporated by reference in its entirety. As another non-limitingexample, the polymer nanoparticle for oral and parenteral formulationsmay be made by the methods described in European Patent No. EP2073848B1,the contents of which are herein incorporated by reference in itsentirety.

In another embodiment, the RNA vaccines described herein may beformulated in nanoparticles used in imaging. The nanoparticles may beliposome nanoparticles such as those described in US Patent PublicationNo US20130129636, herein incorporated by reference in its entirety. As anon-limiting example, the liposome may comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see e.g., USPatent Publication No US20130129636, the contents of which is hereinincorporated by reference in its entirety).

In some embodiments, the nanoparticles which may be used in the presentinvention are formed by the methods described in U.S. Patent ApplicationNo. US20130130348, the contents of which is herein incorporated byreference in its entirety.

The nanoparticles of the present invention may further include nutrientssuch as, but not limited to, those which deficiencies can lead to healthhazards from anemia to neural tube defects (see e.g, the nanoparticlesdescribed in International Patent Publication No WO2013072929, thecontents of which is herein incorporated by reference in its entirety).As a non-limiting example, the nutrient may be iron in the form offerrous, ferric salts or elemental iron, iodine, folic acid, vitamins ormicronutrients.

In some embodiments, the RNA vaccines of the present invention may beformulated in a swellable nanoparticle. The swellable nanoparticle maybe, but is not limited to, those described in U.S. Pat. No. 8,440,231,the contents of which is herein incorporated by reference in itsentirety. As a non-limiting embodiment, the swellable nanoparticle maybe used for delivery of the RNA vaccines of the present invention to thepulmonary system (see e.g., U.S. Pat. No. 8,440,231, the contents ofwhich is herein incorporated by reference in its entirety).

The RNA vaccines of the present invention may be formulated inpolyanhydride nanoparticles such as, but not limited to, those describedin U.S. Pat. No. 8,449,916, the contents of which is herein incorporatedby reference in its entirety.

The nanoparticles and microparticles of the present invention may begeometrically engineered to modulate macrophage and/or the immuneresponse. In one aspect, the geometrically engineered particles may havevaried shapes, sizes and/or surface charges in order to incorporated thepolynucleotides of the present invention for targeted delivery such as,but not limited to, pulmonary delivery (see e.g., InternationalPublication No WO2013082111, the contents of which is hereinincorporated by reference in its entirety). Other physical features thegeometrically engineering particles may have include, but are notlimited to, fenestrations, angled arms, asymmetry and surface roughness,charge which can alter the interactions with cells and tissues. As anon-limiting example, nanoparticles of the present invention may be madeby the methods described in International Publication No WO2013082111,the contents of which is herein incorporated by reference in itsentirety.

In some embodiments, the nanoparticles of the present invention may bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013090601, the contents of which isherein incorporated by reference in its entirety. The nanoparticles maybe inorganic nanoparticles which have a compact and zwitterionic ligandin order to exhibit good water solubility. The nanoparticles may alsohave small hydrodynamic diameters (HD), stability with respect to time,pH, and salinity and a low level of non-specific protein binding.

In some embodiments the nanoparticles of the present invention may bedeveloped by the methods described in US Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the nanoparticles of the present invention arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in US Patent Publication No.US20130172406; the contents of which is herein incorporated by referencein its entirety. The nanoparticles of the present invention may be madeby the methods described in US Patent Publication No. US20130172406, thecontents of which are herein incorporated by reference in its entirety.

In another embodiment, the stealth or target-specific stealthnanoparticles may comprise a polymeric matrix. The polymeric matrix maycomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In some embodiments, the nanoparticle may be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure maymade by the methods described in US Patent Publication No.US20130171646, the contents of which are herein incorporated byreference in its entirety. The nanoparticle may comprise a nucleic acidsuch as, but not limited to, polynucleotides described herein and/orknown in the art.

At least one of the nanoparticles of the present invention may beembedded in in the core a nanostructure or coated with a low densityporous 3-D structure or coating which is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure. Non-limiting examples of the nanostructures comprising atleast one nanoparticle are described in International Patent PublicationNo. WO2013123523, the contents of which are herein incorporated byreference in its entirety.

In some embodiments the RNA (e.g., mRNA) vaccine may be associated witha cationic or polycationic compounds, including protamine, nucleoline,spermine or spermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP²² derived or analog peptides,Pestivirus Ems, HSV, VP²² (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs), PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,Calcitonin peptide(s), Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan,Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,histones, cationic polysaccharides, for example chitosan, polybrene,cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP,DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC,DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolaminechloride, CLIP 1:rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo-nium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole), etc.

In other embodiments the RNA (e.g., mRNA) vaccine is not associated witha cationic or polycationic compounds.

Multimeric Complexes

The RNA vaccines described herein can be assembled as multimericcomplexes having non-covalent (e.g., hydrogen bonds) linkages betweenmRNA molecules. These types of multimeric structures allow for uniformdistribution of the mRNA in a therapeutic composition. When multiplenucleic acids such as RNA are formulated, for instance, in a lipid basedformulation, a relatively uniform distribution of the total nucleic acidthrough the formulation may be achieved. However, the distribution of aparticular nucleic acid with respect to the other nucleic acids in themixture is not uniform. For instance when the nucleic acid mixture iscomposed of two distinct mRNA sequences, some of the lipid particles orother formulatory agents will house a single mRNA sequence, while otherswill house the other mRNA sequence and a few will house both of the mRNAsequences. In a therapeutic context this uneven distribution of mRNA isundesirable because the dosage of the mRNA being delivered to a patientwill vary from administration to administration. Quite surprisingly, themultimeric structures described herein have enabled the production offormulations having nucleic acids with a uniform distribution throughoutthe formulation. It was surprising that a non-covalent interactionbetween the individual nucleic acids would be capable of producing sucha uniform distribution of the nucleic acids in a formulation.Additionally, the multimeric nucleic acid complexes do not interferewith activity such as mRNA expression activity.

In some embodiments the multimeric structures of the RNA polynucleotidesmaking up the vaccine are uniformly distributed throughout a compositionsuch as a lipid nanoparticle. Uniformly distributed, as used herein inthe context of multiple nucleic acids (each having a unique nucleotidesequence), refers to the distribution of each of the nucleic acidsrelative to one another in the formulation. Distribution of the nucleicacids in a formulation may be assessed using methods known in the art. Anucleic acid is uniformly distributed relative to another nucleic acidif the nucleic acid is associated in proximity within a particular areaof the formulation to the other nucleic acid at an approximately 1:1ratio. In some embodiments the nucleic acid is uniformly distributedrelative to another nucleic acid if the nucleic acid is positionedwithin a particular area of the formulation to the other nucleic acid atan approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8,1:1.9, or 1:2 ratio.

A multimeric structure as used herein is series of at least nucleicacids linked together to form a multimeric structure. In someembodiments a multimeric structure is composed of 2 or more, 3 or more,4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more nucleicacids. In other embodiments the multimeric structure is composed of 1000or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less,40 or less, 30 or less, 20 or less or 100 or less nucleic acids. In yetother embodiments a multimeric structure has 3-100, 5-100, 10-100,15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100,60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15-50,20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300,100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 nucleic acids.In preferred embodiments a multimeric structure is composed of 3-5nucleic acids.

In some embodiments the upper limit on the number of nucleic acids in amultimeric structure depends on the length of dimerizable region. Agreater than 20-nucleotide space between mRNAs can provide specificityand enough force to keep the multi-mRNA complex intact for downstreamprocessing and is thus preferred in some embodiments. In someembodiments 4-5 nucleic acids in a multimeric structure may be desirablefor vaccines.

The multimeric structures may be self-assembling multimeric mRNAstructures composed of a first mRNA having a first linking regioncomprised of a part A and a part B and a second mRNA having a secondlinking region comprised of a part C and a part D, wherein at least partA of the first and at least part C of the second linking regions arecomplementary to one another. Preferably the nucleic acids are linked toone another through a non-covalent bond in the linking regions.

A linking region, as used herein, refers to a nucleic acid sequencehaving one or more regions or parts that are complementary to one ormore regions of other linking regions. A pair of linking regions, eachhaving one complementary region, may be at least 70% complementary toone another. In some embodiments a pair of linking regions are at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to oneanother. A linking region may be composed of sub-parts, optionallyreferred to as parts A, B, C, D, . . . , which have shorter regions ofcomplementarity between one another, such that the subparts may becomplementary with other sub-parts. For instance, a simple multimericstructure of two mRNAs can each have a linking region with a singleregion of complementarity. The two linking regions are able to formnon-covalent interactions with one another through base pairing. Morecomplex multimeric structures are also contemplated wherein a linkingregion of each nucleic acid has at least two parts, each part havingcomplementarity with a part on another nucleic acid linking region.Linking regions having multiple parts with different complementarityenables the production of larger multimeric complexes of 3, 4, 5 or morenucleic acids.

The linking regions in some embodiments are 5-100 nucleotides in length.In other embodiments the linking regions are 10-25 nucleotides inlength.

As used herein, the term “region of complementarity” refers to a regionon a first nucleic acid strand that is substantially complementary to asecond region on a second nucleic acid strand. Generally, two nucleicacids sharing a region of complementarity are capable, under suitableconditions, of hybridizing (e.g., via nucleic acid base pairing) to forma duplex structure. A region of complementarity can vary in size. Insome embodiments, a region of complementarity ranges in length fromabout 2 base pairs to about 100 base pairs. In some embodiments, aregion of complementarity ranges in length from about 5 base pairs toabout 75 base pairs. In some embodiments, a region of complementarityranges in length from about 10 base pairs to about 50 base pairs. Insome embodiments, a region of complementarity ranges in length fromabout 20 base pairs to about 30 base pairs.

The number of nucleic acid bases shared between two nucleic acids acrossa region of complementarity can vary. In some embodiments, two nucleicacids share 100% complementary base pairs (e.g., no mismatches) across aregion of complementarity. In some embodiments, two nucleic acids shareat least 99.9%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75% or at least 70% complementary base pairs across a region ofcomplementarity. In some embodiments, a region of complementarity sharedbetween two nucleic acids includes at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 base pair mismatches. In some embodiments, a region ofcomplementarity shared between two nucleic acids includes more than 10base pair mismatches.

As used herein, the term “non-covalent bond” refers to a chemicalinteraction (e.g., joining) between molecules that does not involve thesharing of electrons. Generally, non-covalent bonds are formed viaelectromagnetic interactions between charged molecules. Examples ofnon-covalent bonds include, but are not limited to, ionic bonds,hydrogen bonds, halogen bonds, Van der Waals forces (e.g., dipole-dipoleinteractions, London dispersion forces, etc.), π-effects (π-πinteractions, cation-π interactions, anion-π interactions), andhydrophobic effect.

In some embodiments, at least one non-covalent bond formed between thenucleic acid molecules (e.g., mRNA molecules) of a multimeric moleculeis a result of Watson-Crick base-pairing. The term “Watson-Crickbase-pairing”, or “base-pairing” refers to the formation of hydrogenbonds between specific pairs of nucleotide bases (“complementary basepairs”). For example, two hydrogen bonds form between adenine (A) anduracil (U), and three hydrogen bonds form between guanine (G) andcytosine (C). One method of assessing the strength of bonding betweentwo polynucleotides is by quantifying the percentage of bonds formedbetween the guanine and cytosine bases of the two polynucleotides (“GCcontent”). In some embodiments, the GC content of bonding between twonucleic acids of a multimeric molecule (e.g., a multimeric mRNAmolecule) is at least 10%, at least 20%, at least 30%, at least 40%, orat least 50%. In some embodiments, the GC content of bonding between twonucleic acids of a multimeric molecule (e.g., a multimeric mRNAmolecule) is between 10% and 70%, about 20% to about 60%, or about 30%to about 60%. The formation of a nucleic acid duplex via bonding ofcomplementary base pairs can also be referred to as “hybridization”.

In some embodiments, two nucleic acid molecules (e.g., mRNA molecules)hybridize to form a multimeric molecule. Hybridization can result fromthe formation of at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10non-covalent bonds between two polynucleotides (e.g., mRNA molecules).In some embodiments, between about 2 non-covalent bonds and about 10non-covalent bonds are formed between two nucleic acid molecules. Insome embodiments, between about 5 and about 15 non-covalent bonds areformed between two nucleic acid molecules. In some embodiments, betweenabout 10 and about 20 non-covalent bonds are formed between two nucleicacid molecules. In some embodiments, between about 15 and about 30non-covalent bonds are formed between two nucleic acid molecules. Insome embodiments, between about 20 and about 50 non-covalent bonds areformed between two nucleic acid molecules. In some embodiments, thenumber of non-covalent bonds formed between two nucleic acid molecules(e.g., mRNA molecules) is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or 50 non-covalent bonds.

In some embodiments the self-assembling multimeric mRNA structure iscomprised of at least 2-100 mRNAs each mRNA having a linking region anda stabilizing nucleic acid, wherein the stabilizing nucleic acid has anucleotide sequence with regions complementary to each linking region. Astabilizing nucleic acid as used herein is any nucleic acid that hasmultiple linking regions and is capable of forming non-covalentinteractions with at least 2, but more preferably, 3, 4, 5, 6,7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 other nucleic acids. For instance the stabilizingnucleic acid may have the following structure:

L₁X₁L₂X₂L₃X₃L₄X₄L₅X₅L₆X₆ wherein L is a nucleic acid sequencecomplementary to a linking region and wherein x is any nucleic acidsequence 0-50 nucleotides in length.

In some embodiments, a multimeric mRNA molecule comprises a first mRNAand a second mRNA, wherein the first mRNA and the second mRNA arenon-covalently linked to one another through a splint. As used herein,the term “splint” refers to an oligonucleotide having a first region ofcomplementarity with the first nucleic acid and a second region ofcomplementarity with the second nucleic acid. A splint can be a DNAoligonucleotide or an RNA oligonucleotide. In some embodiments, a splintcomprises one or more modified oligonucleotides. In some embodiments, asplint is non-covalently linked to a 5′UTR of an mRNA. In someembodiments, a splint is non-covalently linked to a 3′UTR of an mRNA. Insome embodiments, non-covalent bonds between nucleic acid molecules(e.g., mRNA molecules) are formed in a non-coding region of eachmolecule. As used herein, the term “non-coding region” refers to alocation of a polynucleotide (e.g., an mRNA) that is not translated intoa protein. Examples of non-coding regions include regulatory regions(e.g., DNA binding domains, promoter sequences, enhancer sequences), anduntranslated regions (e.g., 5′UTR, 3′UTR). In some embodiments, thenon-coding region is an untranslated region (UTR).

By definition, wild type untranslated regions (UTRs) of a gene aretranscribed but not translated. In mRNA, the 5′UTR starts at thetranscription start site and continues to the start codon but does notinclude the start codon; whereas, the 3′UTR starts immediately followingthe stop codon and continues until the transcriptional terminationsignal.

Natural 5′UTRs bear features which play roles in translation initiation.They harbor signatures like Kozak sequences which are commonly known tobe involved in the process by which the ribosome initiates translationof many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, whereR is a purine (adenine or guanine) three bases upstream of the startcodon (AUG), which is followed by another ‘G’. 5′UTR also have beenknown to form secondary structures which are involved in elongationfactor binding.

It should be understood that any UTR from any gene may be incorporatedinto the regions of the polynucleotide. Furthermore, multiple wild-typeUTRs of any known gene may be utilized. It is also within the scope ofthe present invention to provide artificial UTRs which are not variantsof wild type regions. These UTRs or portions thereof may be placed inthe same orientation as in the transcript from which they were selectedor may be altered in orientation or location. Hence a 5′ or 3′ UTR maybe inverted, shortened, lengthened, made with one or more other 5′ UTRsor 3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5′ or3′ UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In some embodiments, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new polynucleotide. As used herein, a “family of proteins” isused in the broadest sense to refer to a group of two or morepolypeptides of interest which share at least one function, structure,feature, localization, origin, or expression pattern. The untranslatedregion may also include translation enhancer elements (TEE).

In some embodiments, an UTR of a polynucleotide (e.g., a first nucleicacid) of the present invention is engineered or modified to have regionsof complementarity with an UTR of another polynucleotide (a secondnucleic acid). For example, UTR nucleotide sequences of twopolynucleotides sought to be joined (e.g., in a multimeric molecule) canbe modified to include a region of complementarity such that the twoUTRs hybridize to form a multimeric molecule.

In some embodiments, the 5′UTR of an RNA polynucleotide encoding an HCMVantigenic polypeptide is modified to allow the formation of a multimericsequence. In some embodiments, the 5′UTR of an RNA polynucleotideencoding an HCMV protein selected from gH, gL, gB, gO, gM, gM, UL128,UL130, UL131A1 is modified to allow the formation of a multimericsequence. In some embodiments, the 5′UTR of an RNA polynucleotideencoding an HCMV protein selected from UL128, UL130, UL131A1 is modifiedto allow the formation of a multimeric sequence. In some embodiments,the 5′UTR of an RNA polynucleotide encoding an HCMV glycoprotein ismodified to allow the formation of a multimeric sequence. In someembodiments, the 5′UTR of an RNA polynucleotide encoding an HCMVglycoprotein selected from gH, gL, gB, gO, gM, and gM is modified toallow the formation of a multimeric sequence. In any of theseembodiments, the multimer may be a dimer, a trimer, pentamer, hexamer,heptamer, octamer nonamer, or decamer. In any of these embodiments, themultimer may be a homogenous multimer, that is, it may comprise dimers,trimers, pentamers etc having sequence encoding the same HCMV antigenicpolypeptide. In any of these embodiments, the multimer may be aheterogeneous multimer comprising dimers, trimers, pentamers etc havingsequence encoding different HCMV antigenic polypeptides, for example twodifferent antigenic polypeptides, three different antigenicpolypeptides, four different antigenic polypeptide, five differentantigenic polypeptides, etc. Exemplary HCMV nucleic acids havingmodified 5′UTR sequence for the formation of a multimeric molecule(e.g., dimers, trimers, pentamers, etc) comprise SEQ ID Nos: 19-26.

In some embodiments the RNA vaccine may be associated with a cationic orpolycationic compounds, including protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP²² derived or analog peptides,Pestivirus Ems, HSV, VP²² (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs), PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,Calcitonin peptide(s), Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan,Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,histones, cationic polysaccharides, for example chitosan, polybrene,cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP,DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC,DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolaminechloride, CLIP 1:rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo-nium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

In other embodiments the RNA vaccine is not associated with a cationicor polycationic compounds.

Modes of Vaccine Administration

HCMV RNA vaccines may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited,to intradermal, intramuscular, and/or subcutaneous administration. Thepresent disclosure provides methods comprising administering RNAvaccines to a subject in need thereof. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the disease, the particularcomposition, its mode of administration, its mode of activity, and thelike. HCMV RNA vaccines compositions are typically formulated in dosageunit form for ease of administration and uniformity of dosage.

It will be understood, however, that the total daily usage of HCMV RNAvaccines compositions may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In some embodiments, HCMV RNA vaccines compositions may be administeredat dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one ormore times a day, per week, per month, etc. to obtain the desiredtherapeutic, diagnostic, prophylactic, or imaging effect (see e.g., therange of unit doses described in International Publication NoWO2013078199, herein incorporated by reference in its entirety). Thedesired dosage may be delivered three times a day, two times a day, oncea day, every other day, every third day, every week, every two weeks,every three weeks, every four weeks, every 2 months, every three months,every 6 months, etc. In certain embodiments, the desired dosage may bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, HCMV RNA vaccines compositions may be administered atdosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g.,about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg,about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004mg/kg or about 0.005 mg/kg.

In some embodiments, HCMV RNA vaccine compositions may be administeredonce or twice (or more) at dosage levels sufficient to deliver 0.025mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, HCMV RNA vaccine compositions may be administeredtwice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later,or Day 0 and 10 years later) at a total dose of or at dosage levelssufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg,0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg,0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg,0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg,0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg,0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg,0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency ofadministration are encompassed by the present disclosure. For example, aHCMV RNA vaccine composition may be administered three or four times.

In some embodiments, HCMV RNA vaccine compositions may be administeredtwice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later,or Day 0 and 10 years later) at a total dose of or at dosage levelssufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or0.400 mg.

In some embodiments the RNA vaccine for use in a method of vaccinating asubject is administered to the subject in a single dosage of between 10μg/kg and 400 μg/kg of the nucleic acid vaccine in an effective amountto vaccinate the subject. In some embodiments the RNA vaccine for use ina method of vaccinating a subject is administered to the subject in asingle dosage of between 10 μg and 400 μg of the nucleic acid vaccine inan effective amount to vaccinate the subject. In some embodiments, anHCMV RNA (e.g., mRNA) vaccine for use in a method of vaccinating asubject is administered to the subject in a single dosage of 10 μg. Insome embodiments, an HCMV RNA vaccine for use in a method of vaccinatinga subject is administered to the subject in a single dosage of 2 μg. Insome embodiments, an HCMV RNA vaccine for use in a method of vaccinatinga subject is administered to the subject in two dosages of 10 μg. Insome embodiments, an HCMV RNA vaccine for use in a method of vaccinatinga subject is administered the subject two dosages of 2 μg.

HCMV vaccines described herein can contain multiple RNA polynucleotides.The RNA polynucleotides can be present in equal or different amountswithin the vaccine. For example, a vaccine can comprise: an RNApolynucleotide having an open reading frame encoding HCMV antigenicpolypeptide gH, or an antigenic fragment or epitope thereof; an RNApolynucleotide having an open reading frame encoding HCMV antigenicpolypeptide gL, or an antigenic fragment or epitope thereof; an RNApolynucleotide having an open reading frame encoding HCMV antigenicpolypeptide UL128, or an antigenic fragment or epitope thereof; an RNApolynucleotide having an open reading frame encoding HCMV antigenicpolypeptide UL130, or an antigenic fragment or epitope thereof; an RNApolynucleotide having an open reading frame encoding HCMV antigenicpolypeptide UL131A, or an antigenic fragment or epitope thereof; and/oran RNA polynucleotide having an open reading frame encoding HCMVantigenic polypeptide gB, or an antigenic fragment or epitope thereof.In some embodiments, the ratio of gH-gL-UL128-UL130-UL131A isapproximately 1:1:1:1:1. In other embodiments, the ratio ofgH-gL-UL128-UL 130-UL131A is approximately 4:2:1:1:1. In someembodiments, the ratio of gB-gH-gL-UL128-UL130-UL131A is approximately1:1:1:1:1:1. In some embodiments, the vaccine comprises an equimolarconcentration of gH, gL, UL128, UL130, and UL131A. In some embodiments,the vaccine comprises an equimolar concentration of gB, gH, gL, UL128,UL130, and UL131A. In some embodiments, the vaccine comprises an equalmass of gH, gL, UL128, UL130, and UL131A. In some embodiments, thevaccine comprises an equal mass of gB, gH, gL, UL128, UL130, and UL131A.

An HCMV RNA vaccine pharmaceutical composition described herein can beformulated into a dosage form described herein, such as an intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal,and subcutaneous).

HCMV RNA Vaccine Formulations and Methods of Use

Some aspects of the present disclosure provide formulations of the HCMVRNA (e.g., mRNA) vaccine, wherein the HCMV RNA vaccine is formulated inan effective amount to produce an antigen specific immune response in asubject (e.g., production of antibodies specific to an anti-HCMVantigenic polypeptide). “An effective amount” is a dose of an HCMV RNA(e.g., mRNA) vaccine effective to produce an antigen-specific immuneresponse. Also provided herein are methods of inducing anantigen-specific immune response in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-HCMV antigenic polypeptide antibodytiter produced in a subject administered an HCMV RNA (e.g., mRNA)vaccine as provided herein. An antibody titer is a measurement of theamount of antibodies within a subject, for example, antibodies that arespecific to a particular antigen (e.g., an anti-HCMV antigenicpolypeptide) or epitope of an antigen. Antibody titer is typicallyexpressed as the inverse of the greatest dilution that provides apositive result. Enzyme-linked immunosorbent assay (ELISA) is a commonassay for determining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by the HCMV RNAvaccine.

In some embodiments, an anti-HCMV antigenic polypeptide antibody titerproduced in a subject is increased by at least 1 log relative to acontrol. For example, anti-HCMV antigenic polypeptide antibody titerproduced in a subject may be increased by at least 1.5, at least 2, atleast 2.5, or at least 3 log relative to a control. In some embodiments,the anti-HCMV antigenic polypeptide antibody titer produced in thesubject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control.In some embodiments, the anti-HCMV antigenic polypeptide antibody titerproduced in the subject is increased by 1-3 log relative to a control.For example, the anti-HCMV antigenic polypeptide antibody titer producedin a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5,1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-HCMV antigenic polypeptide antibody titerproduced in a subject is increased at least 2 times relative to acontrol. For example, the anti-HCMV antigenic polypeptide antibody titerproduced in a subject may be increased at least 3 times, at least 4times, at least 5 times, at least 6 times, at least 7 times, at least 8times, at least 9 times, or at least 10 times relative to a control. Insome embodiments, the anti-HCMV antigenic polypeptide antibody titerproduced in the subject is increased 2, 3, 4, 5,6, 7, 8, 9, or 10 timesrelative to a control. In some embodiments, the anti-HCMV antigenicpolypeptide antibody titer produced in a subject is increased 2-10 timesrelative to a control. For example, the anti-HCMV antigenic polypeptideantibody titer produced in a subject may be increased 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9,4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

A control, in some embodiments, is the anti-HCMV antigenic polypeptideantibody titer produced in a subject who has not been administered anHCMV RNA (e.g., mRNA) vaccine. In some embodiments, a control is ananti-HCMV antigenic polypeptide antibody titer produced in a subject whohas been administered a live attenuated HCMV vaccine. An attenuatedvaccine is a vaccine produced by reducing the virulence of a viable(live). An attenuated virus is altered in a manner that renders itharmless or less virulent relative to live, unmodified virus. In someembodiments, a control is an anti-HCMV antigenic polypeptide antibodytiter produced in a subject administered inactivated HCMV vaccine. Insome embodiments, a control is an anti-HCMV antigenic polypeptideantibody titer produced in a subject administered a recombinant orpurified HCMV protein vaccine. Recombinant protein vaccines typicallyinclude protein antigens that either have been produced in aheterologous expression system (e.g., bacteria or yeast) or purifiedfrom large amounts of the pathogenic organism.

In some embodiments, an effective amount of an HCMV RNA (e.g., mRNA)vaccine is a dose that is reduced compared to the standard of care doseof a recombinant HCMV protein vaccine. A “standard of care,” as providedherein, refers to a medical or psychological treatment guideline and canbe general or specific. “Standard of care” specifies appropriatetreatment based on scientific evidence and collaboration between medicalprofessionals involved in the treatment of a given condition. It is thediagnostic and treatment process that a physician/clinician shouldfollow for a certain type of patient, illness or clinical circumstance.A “standard of care dose,” as provided herein, refers to the dose of arecombinant or purified HCMV protein vaccine, or a live attenuated orinactivated HCMV vaccine, that a physician/clinician or other medicalprofessional would administer to a subject to treat or prevent HCMV, oran HCMV-related condition, while following the standard of careguideline for treating or preventing HCMV, or an HCMV-related condition.

In some embodiments, the anti-HCMV antigenic polypeptide antibody titerproduced in a subject administered an effective amount of an HCMV RNAvaccine is equivalent to an anti-HCMV antigenic polypeptide antibodytiter produced in a control subject administered a standard of care doseof a recombinant or purified HCMV protein vaccine or a live attenuatedor inactivated HCMV vaccine.

In some embodiments, an effective amount of an HCMV RNA (e.g., mRNA)vaccine is a dose equivalent to an at least 2-fold reduction in astandard of care dose of a recombinant or purified HCMV protein vaccine.For example, an effective amount of an HCMV RNA vaccine may be a doseequivalent to an at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold reduction in a standard of care dose of a recombinant orpurified HCMV protein vaccine. In some embodiments, an effective amountof an HCMV RNA vaccine is a dose equivalent to an at least at least100-fold, at least 500-fold, or at least 1000-fold reduction in astandard of care dose of a recombinant or purified HCMV protein vaccine.In some embodiments, an effective amount of an HCMV RNA vaccine is adose equivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-,100-, 250-, 500-, or 1000-fold reduction in a standard of care dose of arecombinant or purified HCMV protein vaccine. In some embodiments, theanti-HCMV antigenic polypeptide antibody titer produced in a subjectadministered an effective amount of an HCMV RNA vaccine is equivalent toan anti-HCMV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orprotein HCMV protein vaccine or a live attenuated or inactivated HCMVvaccine. In some embodiments, an effective amount of an HCMV RNA (e.g.,mRNA) vaccine is a dose equivalent to a 2-fold to 1000-fold (e.g.,2-fold to 100-fold, 10-fold to 1000-fold) reduction in the standard ofcare dose of a recombinant or purified HCMV protein vaccine, wherein theanti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments, the effective amount of an HCMV RNA (e.g., mRNA)vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to 800-, 2 to700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to 200-, 2 to 100-,2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to 40-, 2 to 30-, 2to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2 to 5-, 2 to 4-,2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3 to 600-, 3 to500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to 90-, 3 to80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to 4to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4to 50-4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to 7-,4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5 to 700-,5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-, 5 to90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-, 5 to20-, 5 to 10-, 5 to 9-,5 to 8-, 5 to 7-, 5 to 6-, 6 to 1000-, 6 to 900-,6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6 to 300-, 6 to200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-, 6 to 50-, 6 to40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to 7-, 7 to1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7 to 400-,7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-, 7 to 60-,7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to 500-, 8 to 400-,8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-,8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-, 9 to50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10 to 900-, 10to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-, 10 to 300-, 10to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10 to 60-, 10 to50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to 900-, 20 to800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to 300-, 20 to200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to 60-, 20 to 50-,20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to 800-, 30 to 700-,30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-,30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-, 30 to 40-, 40 to1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40 to70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50 to 800-, 50 to700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to 200-, 50 to100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to 1000-, 60 to900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to 400-, 60 to300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to 70-, 70 to1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to 500-, 70 to400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to 80-, 80 to1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to 500-, 80 to400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to 400-, 90 to300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-,100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to 300-, 100 to200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to 600-,200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to 900-, 300 to800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-, 400 to 1000-,400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to 500-, 500 to1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-, 600 to 1000-,600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700 to 900-, 700 to800-, 800 to 1000-, 800 to 900-, or 900 to 1000-fold reduction in thestandard of care dose of a recombinant HCMV protein vaccine. In someembodiments, such as the foregoing, the anti-HCMV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-HCMVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified HCMVprotein vaccine or a live attenuated or inactivated HCMV vaccine. Insome embodiments, the effective amount is a dose equivalent to (orequivalent to an at least) 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-,30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-,160-, 170-, 1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-, 270-,280-, 290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-, 390-,400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-, 510-,520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-, 630-,640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-, 750-,760-, 770-, 780-, 790-, 800-, 810-, 820-, 830-, 840-, 850-, 860-, 870-,880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-, 990-,or 1000-fold reduction in the standard of care dose of a recombinantHCMV protein vaccine. In some embodiments, such as the foregoing, ananti-HCMV antigenic polypeptide antibody titer produced in the subjectis equivalent to an anti-HCMV antigenic polypeptide antibody titerproduced in a control subject administered the standard of care dose ofa recombinant or purified HCMV protein vaccine or a live attenuated orinactivated HCMV vaccine.

In some embodiments, the effective amount of an HCMV RNA (e.g., mRNA)vaccine is a total dose of 50-1000 μg. In some embodiments, theeffective amount of an HCMV RNA (e.g., mRNA) vaccine is a total dose of50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200,50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700,60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70,70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200,70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500,80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700,90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900,100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000,200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000,300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900,400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700,500-600, 600-1000, 600-900, 600-900, 600-700, 700-1000, 700-900,700-800, 800-1000, 800-900, or 900-1000 μg. In some embodiments, theeffective amount of an HCMV RNA (e.g., mRNA) vaccine is a total dose of50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950 or 1000 μg. In some embodiments, the effectiveamount is a dose of 25-500 μg administered to the subject a total of twotimes. In some embodiments, the effective amount of an HCMV RNA (e.g.,mRNA) vaccine is a dose of 25-500, 25-400, 25-300, 25-200, 25-100,25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400,100-300, 100-200, 150-500, 150-400, 150-300, 150-200, 200-500, 200-400,200-300, 250-500, 250-400, 250-300, 300-500, 300-400, 350-500, 350-400,400-500 or 450-500 μg administered to the subject a total of two times.In some embodiments, the effective amount of an HCMV RNA (e.g., mRNA)vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400,450, or 500 μg administered to the subject a total of two times.

In some embodiments, the antigen specific immune response induced by theHCMV RNA vaccines in a subject is the production of antibodies specificto an anti-HCMV antigenic polypeptide. In some embodiments, suchantibodies are capable of neutralizing HCMV in an infected host. In someembodiments, the antigen specific immune response induced by the HCMVRNA vaccines in a subject is antigen-specific T-cell response. SuchT-cell response may provide immunity to the immunized animal (e.g., miceor human) against fution HCMV infections.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1: Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand or parts or regions thereof may be accomplished utilizing themethods taught in International Application WO2014/152027 entitled“Manufacturing Methods for Production of RNA Transcripts”, the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalApplication WO2014/152030 and WO2014/152031, each of which isincorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in WO2014/144039, which is incorporated herein byreference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using a procedure selected from the group consisting ofpolynucleotide mapping, reverse transcriptase sequencing, chargedistribution analysis, and detection of RNA impurities, whereincharacterizing comprises determining the RNA transcript sequence,determining the purity of the RNA transcript, or determining the chargeheterogeneity of the RNA transcript. Such methods are taught in, forexample, WO2014/144711 and WO2014/144767, the contents of each of whichis incorporated herein by reference in its entirety.

Example 2: Chimeric Polynucleotide Synthesis Introduction

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a first region or part of 100 nucleotides orless is chemically synthesized with a 5′ monophosphate and terminal3′desOH or blocked OH. If the region is longer than 80 nucleotides, itmay be synthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then it is preferable that such region or part comprise aphosphate-sugar backbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide is made using a series of starting segments.Such segments include:

(a) Capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) 5′ triphosphate segment which may include the coding region of apolypeptide and comprising a normal 3′OH (SEG. 2)

(c) 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated withcordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct is thenpurified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 μM) 0.75 μl; ReversePrimer (10 μM) 0.75 μl; Template cDNA−100 ng; and dH₂0 diluted to 25.0μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then72° C. for 5 min. then 4° C. to termination.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 4: In Vitro Transcription (IVT)

The in vitro transcription reaction generates polynucleotides containinguniformly modified polynucleotides. Such uniformly modifiedpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure. The input nucleotide triphosphate (NTP) mix is madein-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

Template cDNA 1.0 μg 10x transcription buffer (400 mM 2.0 μl Tris-HCl pH8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) Custom NTPs (25 mM each)7.2 μl RNase Inhibitor 20 U T7 RNA polymerase 3000 U dH₂0 Up to 20.0 μl.and Incubation at 37° C. for 3 hr-5 hrs.Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 5: Enzymatic Capping

Capping of a polynucleotide is performed as follows where the mixtureincludes: IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixture isincubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10×Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide is then purified using Ambion's MEGACLEAR™ Kit(Austin, Tex.) following the manufacturer's instructions. Following thecleanup, the RNA is quantified using the NANODROP™ (ThermoFisher,Waltham, Mass.) and analyzed by agarose gel electrophoresis to confirmthe RNA is the proper size and that no degradation of the RNA hasoccurred. The RNA product may also be sequenced by running areverse-transcription-PCR to generate the cDNA for sequencing.

Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence polyA tails of approximately between 40-200 nucleotides, e.g,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe invention.

Example 7: Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 8: Capping Assays Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be transfected into cells at equal concentrations. 6,12, 24 and 36 hours post-transfection the amount of protein secretedinto the culture medium can be assayed by ELISA. Syntheticpolynucleotides that secrete higher levels of protein into the mediumwould correspond to a synthetic polynucleotide with a highertranslationally-competent Cap structure.

Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be compared for purity using denaturing Agarose-Ureagel electrophoresis or HPLC analysis. Polynucleotides with a single,consolidated band by electrophoresis correspond to the higher purityproduct compared to polynucleotides with multiple bands or streakingbands. Synthetic polynucleotides with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure polynucleotide population.

Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be transfected into cells at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Polynucleotides resulting in thesecretion of higher levels of pro-inflammatory cytokines into the mediumwould correspond to a polynucleotides containing an immune-activatingcap structure.

Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be analyzed for capping reaction efficiency by LC-MSafter nuclease treatment. Nuclease treatment of capped polynucleotideswould yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and would correspond to cappingreaction efficiency. The cap structure with higher capping reactionefficiency would have a higher amount of capped product by LC-MS.

Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 10: Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μl) are used for Nanodrop UVabsorbance readings to quantitate the yield of each polynucleotide froman chemical synthesis or in vitro transcription reaction.

Example 11: Formulation of Modified mRNA Using Lipidoids

Polynucleotides are formulated for in vitro experiments by mixing thepolynucleotides with the lipidoid at a set ratio prior to addition tocells. In vivo formulation may require the addition of extra ingredientsto facilitate circulation throughout the body. To test the ability ofthese lipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations may used as astarting point. After formation of the particle, polynucleotide is addedand allowed to integrate with the complex. The encapsulation efficiencyis determined using a standard dye exclusion assays.

Example 12: hCMV Vaccine—hCMV Glycoprotein Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

Each of the sequences described herein encompasses a chemically modifiedsequence or an unmodified sequence which includes no nucleotidemodifications.

5′UTR is bolded 3′UTR is underlinedhCMV-gH: hCMV, glycoprotein H (Merlin Strain)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACAGTCGTCTCCTCATGATGTCCGTCTACGCGCTATCGGCCATCATCGGCATCTATCTGCTCTACCGCATGCTCAAGACATGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 1)hCMV-gH: hCMV, glycoprotein H (Merlin Strain)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACAGUCGUCUCCUCAUGAUGUCCGUCUACGCGCUAUCGGCCAUCAUCGGCAUCUAUCUGCUCUACCGCAUGCUCAAGACAUGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 79)hCMV-gHFLAG, hCMV glycoproteinH-FLAG tagTCAAGCTTTTGGACCCTCGTAGAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACAGTCGTCTCCTCATGATGTCCGTCTACGCGCTATCGGCCATCATCGGCATCTATCTGCTCTACCGCATGCTCAAGACATGCGATTACAAGGACGATGACGATAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 2)hCMV-gHFLAG, hCMV glycoproteinH-FLAG ta2UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACAGUCGUCUCCUCAUGAUGUCCGUCUACGCGCUAUCGGCCAUCAUCGGCAUCUAUCUGCUCUACCGCAUGCUCAAGACAUGCGAUUACAAGGACGAUGACGAUAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 80)hCMV-gL, hCMV glycoprotein LTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTGCCGCCGCCCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCCTCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAGGTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGTTACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCGGATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGCGATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTCACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAACCGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTCTGCCTGCGTCACCAGCTGGACCCGCCGCTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGCGTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 3)hCMV-gL, hCMV glycoprotein LUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGUGCCGCCGCCCGGAUUGCGGCUUCUCUUUCUCACCUGGACCGGUGAUACUGCUGUGGUGUUGCCUUCUGCUGCCCAUUGUUUCCUCAGCCGCCGUCAGCGUCGCUCCUACCGCCGCCGAGAAAGUCCCCGCGGAGUGCCCCGAACUAACGCGCCGAUGCUUGUUGGGUGAGGUGUUUGAGGGUGACAAGUAUGAAAGUUGGCUGCGCCCGUUGGUGAAUGUUACCGGGCGCGAUGGCCCGCUAUCGCAACUUAUCCGUUACCGUCCCGUUACGCCGGAGGCCGCCAACUCCGUGCUGUUGGACGAGGCUUUCCUGGACACUCUGGCCCUGCUGUACAACAAUCCGGAUCAAUUGCGGGCCCUGCUGACGCUGUUGAGCUCGGACACAGCGCCGCGCUGGAUGACGGUGAUGCGCGGCUACAGCGAGUGCGGCGAUGGCUCGCCGGCCGUGUACACGUGCGUGGACGACCUGUGCCGCGGCUACGACCUCACGCGACUGUCAUACGGGCGCAGCAUCUUCACGGAACACGUGUUAGGCUUCGAGCUGGUGCCACCGUCUCUCUUUAACGUGGUGGUGGCCAUACGCAACGAAGCCACGCGUACCAACCGCGCCGUGCGUCUGCCCGUGAGCACCGCUGCCGCGCCCGAGGGCAUCACGCUCUUUUACGGCCUGUACAACGCAGUGAAGGAAUUCUGCCUGCGUCACCAGCUGGACCCGCCGCUGCUACGCCACCUAGAUAAAUACUACGCCGGACUGCCGCCCGAGCUGAAGCAGACGCGCGUCAACCUGCCGGCUCACUCGCGCUAUGGCCCUCAAGCAGUGGAUGCUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 81)hCMV-gLFLAG, glycoprotein L-FLAGTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTGCCGCCGCCCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCCTCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAGGTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGTTACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCGGATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGCGATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTCACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAACCGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTCTGCCTGCGTCACCAGCTGGACCCGCCGCTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGCGTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCGATTACAAGGACGATGACGATAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 4) hCMV-gLFLAG, glycoprotein L-FLAGUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGUGCCGCCGCCCGGAUUGCGGCUUCUCUUUCUCACCUGGACCGGUGAUACUGCUGUGGUGUUGCCUUCUGCUGCCCAUUGUUUCCUCAGCCGCCGUCAGCGUCGCUCCUACCGCCGCCGAGAAAGUCCCCGCGGAGUGCCCCGAACUAACGCGCCGAUGCUUGUUGGGUGAGGUGUUUGAGGGUGACAAGUAUGAAAGUUGGCUGCGCCCGUUGGUGAAUGUUACCGGGCGCGAUGGCCCGCUAUCGCAACUUAUCCGUUACCGUCCCGUUACGCCGGAGGCCGCCAACUCCGUGCUGUUGGACGAGGCUUUCCUGGACACUCUGGCCCUGCUGUACAACAAUCCGGAUCAAUUGCGGGCCCUGCUGACGCUGUUGAGCUCGGACACAGCGCCGCGCUGGAUGACGGUGAUGCGCGGCUACAGCGAGUGCGGCGAUGGCUCGCCGGCCGUGUACACGUGCGUGGACGACCUGUGCCGCGGCUACGACCUCACGCGACUGUCAUACGGGCGCAGCAUCUUCACGGAACACGUGUUAGGCUUCGAGCUGGUGCCACCGUCUCUCUUUAACGUGGUGGUGGCCAUACGCAACGAAGCCACGCGUACCAACCGCGCCGUGCGUCUGCCCGUGAGCACCGCUGCCGCGCCCGAGGGCAUCACGCUCUUUUACGGCCUGUACAACGCAGUGAAGGAAUUCUGCCUGCGUCACCAGCUGGACCCGCCGCUGCUACGCCACCUAGAUAAAUACUACGCCGGACUGCCGCCCGAGCUGAAGCAGACGCGCGUCAACCUGCCGGCUCACUCGCGCUAUGGCCCUCAAGCAGUGGAUGCUCGCGAUUACAAGGACGAUGACGAUAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 82) hCMV gB, hCMV glycoprotein BTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGATCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGCGGGTAAAGTACGTGGAGGACAAGGTAGTCGACCCGCTACCGCCCTACCTCAAGGGTCTGGACGACCTCATGAGCGGCCTGGGCGCCGCGGGAAAGGCCGTTGGCGTAGCCATTGGGGCCGTGGGTGGCGCGGTGGCCTCCGTGGTCGAAGGCGTTGCCACCTTCCTCAAAAACCCCTTCGGAGCGTTCACCATCATCCTCGTGGCCATAGCTGTAGTCATTATCACTTATTTGATCTATACTCGACAGCGGCGTTTGTGCACGCAGCCGCTGCAGAACCTCTTTCCCTATCTGGTGTCCGCCGACGGGACCACCGTGACGTCGGGCAGCACCAAAGACACGTCGTTACAGGCTCCGCCTTCCTACGAGGAAAGTGTTTATAATTCTGGTCGCAAAGGACCGGGACCACCGTCGTCTGATGCATCCACGGCGGCTCCGCCTTACACCAACGAGCAGGCTTACCAGATGCTTCTGGCCCTGGCCCGTCTGGACGCAGAGCAGCGAGCGCAGCAGAACGGTACAGATTCTTTGGACGGACGGACTGGCACGCAGGACAAGGGACAGAAGCCCAACCTACTAGACCGACTGCGACATCGCAAAAACGGCTACCGACACTTGAAAGACTCTGACGAAGAAGAGAACGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 5) hCMV gB, hCMV glycoprotein BUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCAGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGCGGGUAAAGUACGUGGAGGACAAGGUAGUCGACCCGCUACCGCCCUACCUCAAGGGUCUGGACGACCUCAUGAGCGGCCUGGGCGCCGCGGGAAAGGCCGUUGGCGUAGCCAUUGGGGCCGUGGGUGGCGCGGUGGCCUCCGUGGUCGAAGGCGUUGCCACCUUCCUCAAAAACCCCUUCGGAGCGUUCACCAUCAUCCUCGUGGCCAUAGCUGUAGUCAUUAUCACUUAUUUGAUCUAUACUCGACAGCGGCGUUUGUGCACGCAGCCGCUGCAGAACCUCUUUCCCUAUCUGGUGUCCGCCGACGGGACCACCGUGACGUCGGGCAGCACCAAAGACACGUCGUUACAGGCUCCGCCUUCCUACGAGGAAAGUGUUUAUAAUUCUGGUCGCAAAGGACCGGGACCACCGUCGUCUGAUGCAUCCACGGCGGCUCCGCCUUACACCAACGAGCAGGCUUACCAGAUGCUUCUGGCCCUGGCCCGUCUGGACGCAGAGCAGCGAGCGCAGCAGAACGGUACAGAUUCUUUGGACGGACGGACUGGCACGCAGGACAAGGGACAGAAGCCCAACCUACUAGACCGACUGCGACAUCGCAAAAACGGCUACCGACACUUGAAAGACUCUGACGAAGAAGAGAACGUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 83) hCMV gBFLAG, hCMV glycoproteinB-FLAGTCAAGCTTTTGGACCCTCGTAGAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGATCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGCGGGTAAAGTACGTGGAGGACAAGGTAGTCGACCCGCTACCGCCCTACCTCAAGGGTCTGGACGACCTCATGAGCGGCCTGGGCGCCGCGGGAAAGGCCGTTGGCGTAGCCATTGGGGCCGTGGGTGGCGCGGTGGCCTCCGTGGTCGAAGGCGTTGCCACCTTCCTCAAAAACCCCTTCGGAGCGTTCACCATCATCCTCGTGGCCATAGCTGTAGTCATTATCACTTATTTGATCTATACTCGACAGCGGCGTTTGTGCACGCAGCCGCTGCAGAACCTCTTTCCCTATCTGGTGTCCGCCGACGGGACCACCGTGACGTCGGGCAGCACCAAAGACACGTCGTTACAGGCTCCGCCTTCCTACGAGGAAAGTGTTTATAATTCTGGTCGCAAAGGACCGGGACCACCGTCGTCTGATGCATCCACGGCGGCTCCGCCTTACACCAACGAGCAGGCTTACCAGATGCTTCTGGCCCTGGCCCGTCTGGACGCAGAGCAGCGAGCGCAGCAGAACGGTACAGATTCTTTGGACGGACGGACTGGCACGCAGGACAAGGGACAGAAGCCCAACCTACTAGACCGACTGCGACATCGCAAAAACGGCTACCGACACTTGAAAGACTCTGACGAAGAAGAGAACGTCGATTACAAGGACGATGACGATAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 6)hCMV gBFLAG, hCMV glycoproteinB-FLAGTCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCAGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGCGGGUAAAGUACGUGGAGGACAAGGUAGUCGACCCGCUACCGCCCUACCUCAAGGGUCUGGACGACCUCAUGAGCGGCCUGGGCGCCGCGGGAAAGGCCGUUGGCGUAGCCAUUGGGGCCGUGGGUGGCGCGGUGGCCUCCGUGGUCGAAGGCGUUGCCACCUUCCUCAAAAACCCCUUCGGAGCGUUCACCAUCAUCCUCGUGGCCAUAGCUGUAGUCAUUAUCACUUAUUUGAUCUAUACUCGACAGCGGCGUUUGUGCACGCAGCCGCUGCAGAACCUCUUUCCCUAUCUGGUGUCCGCCGACGGGACCACCGUGACGUCGGGCAGCACCAAAGACACGUCGUUACAGGCUCCGCCUUCCUACGAGGAAAGUGUUUAUAAUUCUGGUCGCAAAGGACCGGGACCACCGUCGUCUGAUGCAUCCACGGCGGCUCCGCCUUACACCAACGAGCAGGCUUACCAGAUGCUUCUGGCCCUGGCCCGUCUGGACGCAGAGCAGCGAGCGCAGCAGAACGGUACAGAUUCUUUGGACGGACGGACUGGCACGCAGGACAAGGGACAGAAGCCCAACCUACUAGACCGACUGCGACAUCGCAAAAACGGCUACCGACACUUGAAAGACUCUGACGAAGAAGAGAACGUCGAUUACAAGGACGAUGACGAUAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 84)

Example 13: hCMV Vaccine—hCMV Variant Glycoprotein Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

hCMV-gHtrunc, hCMV glycoproteinH (Ectodomain)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(SEQ ID NO: 7) hCMV-gHtrunc, hCMV glycoproteinH (Ectodomain)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO: 85) hCMV-gHtruncFLAG, glycoprotein H EctodomainTCAAGCTTTTGGACCCTCGTAGAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACGATTACAAGGACGATGACGATAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 8)hCMV-gHtruncFLAG, glycoprotein H EctodomainUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACGAUUACAAGGACGAUGACGAUAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 86)hCMVgHtrunc6XHis, glycoprotein H Ectodomain-6XHis tagTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACCACCATCACCACCATCACTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 9)hCMVgHtrunc6XHis, glycoprotein H Ectodomain-6XHis tagUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACCACCAUCACCACCAUCACUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 87)hCMV TrgB, glycoprotein B (ectodomain)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGATCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 10)hCMV TrgB, glycoprotein B (ectodomain)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCAGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 88)hCMV TrgBFLAG, hCMV glycoproteinB ectodomain-FLAGTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGATCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGGATTACAAGGACGATGACGATAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 11)hCMV TrgBFLAG, hCMV glycoproteinB ectodomain-FLAGUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCAGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGGAUUACAAGGACGAUGACGAUAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 89)hCMV-TrgB6XHis, hCMV glycoprotein ectodomain-6XHis tagTCAAGCTTTTGGACCCTCGTAGAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGATCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGCACCATCACCACCATCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 12)hCMV-TrgB6XHis, hCMV glycoprotein ectodomain-6XHis tapUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCAGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGCACCAUCACCACCAUCACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 90)

Example 14: hCMV Vaccine—hCMV UL Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

hCMV UL128 (SEQ ID NO: 13)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTCCCAAAGATCTGACGCCGTTCTTGACGGCGTTGTGGCTGCTATTGGGTCACAGCCGCGTGCCGCGGGTGCGCGCAGAAGAATGTTGCGAATTCATAAACGTCAACCACCCGCCGGAACGCTGTTACGATTTCAAAATGTGCAATCGCTTCACCGTCGCGCTGCGGTGTCCGGACGGCGAAGTCTGCTACAGTCCCGAGAAAACGGCTGAGATTCGCGGGATCGTCACCACCATGACCCATTCATTGACACGCCAGGTCGTACACAACAAACTGACGAGCTGCAACTACAATCCGTTATACCTCGAAGCTGACGGGCGAATACGCTGCGGCAAAGTAAACGACAAGGCGCAGTACCTGCTGGGCGCCGCTGGCAGCGTTCCCTATCGATGGATCAATCTGGAATACGACAAGATAACCCGGATCGTGGGCCTGGATCAGTACCTGGAGAGCGTTAAGAAACACAAACGGCTGGATGTGTGCCGCGCTAAAATGGGCTATATGCTGCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV UL128 (SEQ ID NO: 91)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAGUCCCAAAGAUCUGACGCCGUUCUUGACGGCGUUGUGGCUGCUAUUGGGUCACAGCCGCGUGCCGCGGGUGCGCGCAGAAGAAUGUUGCGAAUUCAUAAACGUCAACCACCCGCCGGAACGCUGUUACGAUUUCAAAAUGUGCAAUCGCUUCACCGUCGCGCUGCGGUGUCCGGACGGCGAAGUCUGCUACAGUCCCGAGAAAACGGCUGAGAUUCGCGGGAUCGUCACCACCAUGACCCAUUCAUUGACACGCCAGGUCGUACACAACAAACUGACGAGCUGCAACUACAAUCCGUUAUACCUCGAAGCUGACGGGCGAAUACGCUGCGGCAAAGUAAACGACAAGGCGCAGUACCUGCUGGGCGCCGCUGGCAGCGUUCCCUAUCGAUGGAUCAAUCUGGAAUACGACAAGAUAACCCGGAUCGUGGGCCUGGAUCAGUACCUGGAGAGCGUUAAGAAACACAAACGGCUGGAUGUGUGCCGCGCUAAAAUGGGCUAUAUGCUGCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-128FLAG, UL128-FLAG tag(SEQ ID NO: 14)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTCCCAAAGATCTGACGCCGTTCTTGACGGCGTTGTGGCTGCTATTGGGTCACAGCCGCGTGCCGCGGGTGCGCGCAGAAGAATGTTGCGAATTCATAAACGTCAACCACCCGCCGGAACGCTGTTACGATTTCAAAATGTGCAATCGCTTCACCGTCGCGCTGCGGTGTCCGGACGGCGAAGTCTGCTACAGTCCCGAGAAAACGGCTGAGATTCGCGGGATCGTCACCACCATGACCCATTCATTGACACGCCAGGTCGTACACAACAAACTGACGAGCTGCAACTACAATCCGTTATACCTCGAAGCTGACGGGCGAATACGCTGCGGCAAAGTAAACGACAAGGCGCAGTACCTGCTGGGCGCCGCTGGCAGCGTTCCCTATCGATGGATCAATCTGGAATACGACAAGATAACCCGGATCGTGGGCCTGGATCAGTACCTGGAGAGCGTTAAGAAACACAAACGGCTGGATGTGTGCCGCGCTAAAATGGGCTATATGCTGCAGGATTACAAGGACGATGACGATAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGG CGGChCMV-128FLAG, UL128-FLAG tag (SEQ ID NO: 92)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAGUCCCAAAGAUCUGACGCCGUUCUUGACGGCGUUGUGGCUGCUAUUGGGUCACAGCCGCGUGCCGCGGGUGCGCGCAGAAGAAUGUUGCGAAUUCAUAAACGUCAACCACCCGCCGGAACGCUGUUACGAUUUCAAAAUGUGCAAUCGCUUCACCGUCGCGCUGCGGUGUCCGGACGGCGAAGUCUGCUACAGUCCCGAGAAAACGGCUGAGAUUCGCGGGAUCGUCACCACCAUGACCCAUUCAUUGACACGCCAGGUCGUACACAACAAACUGACGAGCUGCAACUACAAUCCGUUAUACCUCGAAGCUGACGGGCGAAUACGCUGCGGCAAAGUAAACGACAAGGCGCAGUACCUGCUGGGCGCCGCUGGCAGCGUUCCCUAUCGAUGGAUCAAUCUGGAAUACGACAAGAUAACCCGGAUCGUGGGCCUGGAUCAGUACCUGGAGAGCGUUAAGAAACACAAACGGCUGGAUGUGUGCCGCGCUAAAAUGGGCUAUAUGCUGCAGGAUUACAAGGACGAUGACGAUAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGChCMV-UL130 (SEQ ID NO: 15)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCTGCGGCTTCTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCCTGTCTGGCGTCTCCGTGGTCGACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCTTTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACCCTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGTCGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAGATTTTTGGAGCGCACATGGTGCCCAAGCAGACCAAGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATGAAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACTTACACCTTCTGCACCCATCCCAATCTCATCGTTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UL130(SEQ ID NO: 93)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCUGCGGCUUCUGCUUCGUCACCACUUUCACUGCCUGCUUCUGUGCGCGGUUUGGGCAACGCCCUGUCUGGCGUCUCCGUGGUCGACGCUAACAGCAAACCAGAAUCCGUCCCCGCCAUGGUCUAAACUGACGUAUUCCAAACCGCAUGACGCGGCGACGUUUUACUGUCCUUUUCUCUAUCCCUCGCCCCCACGAUCCCCCUUGCAAUUCUCGGGGUUCCAGCGGGUAUCAACGGGUCCCGAGUGUCGCAACGAGACCCUGUAUCUGCUGUACAACCGGGAAGGCCAGACCUUGGUGGAGAGAAGCUCCACCUGGGUGAAAAAGGUGAUCUGGUACCUGAGCGGUCGGAACCAAACCAUCCUCCAACGGAUGCCCCGAACGGCUUCGAAACCGAGCGACGGAAACGUGCAGAUCAGCGUGGAAGACGCCAAGAUUUUUGGAGCGCACAUGGUGCCCAAGCAGACCAAGCUGCUACGCUUCGUCGUCAACGAUGGCACACGUUAUCAGAUGUGUGUGAUGAAGCUGGAGAGCUGGGCUCACGUCUUCCGGGACUACAGCGUGUCUUUUCAGGUGCGAUUGACGUUCACCGAGGCCAAUAACCAGACUUACACCUUCUGCACCCAUCCCAAUCUCAUCGUUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGChCMV-UL130FLAG, UL130-FLAG tag (SEQ ID NO: 16)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCTGCGGCTTCTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCCTGTCTGGCGTCTCCGTGGTCGACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCTTTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACCCTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGTCGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAGATTTTTGGAGCGCACATGGTGCCCAAGCAGACCAAGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATGAAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACTTACACCTTCTGCACCCATCCCAATCTCATCGTTGATTACAAGGACGATGACGATAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UL130FLAG, UL130-FLAG tag (SEQ ID NO: 94)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCUGCGGCUUCUGCUUCGUCACCACUUUCACUGCCUGCUUCUGUGCGCGGUUUGGGCAACGCCCUGUCUGGCGUCUCCGUGGUCGACGCUAACAGCAAACCAGAAUCCGUCCCCGCCAUGGUCUAAACUGACGUAUUCCAAACCGCAUGACGCGGCGACGUUUUACUGUCCUUUUCUCUAUCCCUCGCCCCCACGAUCCCCCUUGCAAUUCUCGGGGUUCCAGCGGGUAUCAACGGGUCCCGAGUGUCGCAACGAGACCCUGUAUCUGCUGUACAACCGGGAAGGCCAGACCUUGGUGGAGAGAAGCUCCACCUGGGUGAAAAAGGUGAUCUGGUACCUGAGCGGUCGGAACCAAACCAUCCUCCAACGGAUGCCCCGAACGGCUUCGAAACCGAGCGACGGAAACGUGCAGAUCAGCGUGGAAGACGCCAAGAUUUUUGGAGCGCACAUGGUGCCCAAGCAGACCAAGCUGCUACGCUUCGUCGUCAACGAUGGCACACGUUAUCAGAUGUGUGUGAUGAAGCUGGAGAGCUGGGCUCACGUCUUCCGGGACUACAGCGUGUCUUUUCAGGUGCGAUUGACGUUCACCGAGGCCAAUAACCAGACUUACACCUUCUGCACCCAUCCCAAUCUCAUCGUUGAUUACAAGGACGAUGACGAUAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-UL131A (SEQ ID NO: 17)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCTGTGTCGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGGAAACCGCGGAAAAAAACGATTATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGCCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGACCTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGAATCAACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCCGCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UL131A (SEQ ID NO: 95)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCUGUGUCGGGUGUGGCUGUCUGUUUGUCUGUGCGCCGUGGUGCUGGGUCAGUGCCAGCGGGAAACCGCGGAAAAAAACGAUUAUUACCGAGUACCGCAUUACUGGGACGCGUGCUCUCGCGCGCUGCCCGACCAAACCCGUUACAAGUAUGUGGAACAGCUCGUGGACCUCACGUUGAACUACCACUACGAUGCGAGCCACGGCUUGGACAACUUUGACGUGCUCAAGAGAAUCAACGUGACCGAGGUGUCGUUGCUCAUCAGCGACUUUAGACGUCAGAACCGUCGCGGCGGCACCAACAAAAGGACCACGUUCAACGCCGCCGGUUCGCUGGCGCCACACGCCCGGAGCCUCGAGUUCAGCGUGCGGCUCUUUGCCAACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-UL131AFLAG, UL131A-FLAG(SEQ ID NO: 18)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCTGTGTCGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGGAAACCGCGGAAAAAAACGATTATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGCCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGACCTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGAATCAACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCCGCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACGATTACAAGGACGATGACGATAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGG GCGGChCMV-UL131AFLAG, UL131A-FLAG (SEQ ID NO: 96)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCUGUGUCGGGUGUGGCUGUCUGUUUGUCUGUGCGCCGUGGUGCUGGGUCAGUGCCAGCGGGAAACCGCGGAAAAAAACGAUUAUUACCGAGUACCGCAUUACUGGGACGCGUGCUCUCGCGCGCUGCCCGACCAAACCCGUUACAAGUAUGUGGAACAGCUCGUGGACCUCACGUUGAACUACCACUACGAUGCGAGCCACGGCUUGGACAACUUUGACGUGCUCAAGAGAAUCAACGUGACCGAGGUGUCGUUGCUCAUCAGCGACUUUAGACGUCAGAACCGUCGCGGCGGCACCAACAAAAGGACCACGUUCAACGCCGCCGGUUCGCUGGCGCCACACGCCCGGAGCCUCGAGUUCAGCGUGCGGCUCUUUGCCAACGAUUACAAGGACGAUGACGAUAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC

Example 15: hCMV Vaccine—hCMV UL Multimeric Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

hCMV gH penta (SEQ ID NO: 19)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGACAGACGAGAGAGAAGCACGCCAATTCTGCCTGCTTAAGCCATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACAGTCGTCTCCTCATGATGTCCGTCTACGCGCTATCGGCCATCATCGGCATCTATCTGCTCTACCGCATGCTCAAGACATGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV gH penta (SEQ ID NO: 97)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGACAGACGAGAGAGAAGCACGCCAAUUCUGCCUGCUUAAGCCAUGCGGCCAGGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACUUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUUUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUUUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUUUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAAAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAAAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACAGUCGUCUCCUCAUGAUGUCCGUCUACGCGCUAUCGGCCAUCAUCGGCAUCUAUCUGCUCUACCGCAUGCUCAAGACAUGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV gL penta (SEQ ID NO: 20)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGCTTAAGCAGGCAGAATTGGCCCTTAGCCTGTACCAGCCGAACCATGTGCCGCCGCCCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCCTCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAGGTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGTTACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCGGATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGCGATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTCACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAACCGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTCTGCCTGCGTCACCAGCTGGACCCGCCGGTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGCGTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV gL penta(SEQ ID NO: 98)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGCUUAAGCAGGCAGAAUUGGCCCUUAGCCUGUACCAGCCGAACCAUGUGCCGCCGCCCGGAUUGCGGCUUCUCUUUCUCACCUGGACCGGUGAUACUGCUGUGGUGUUGCCUUCUGCUGCCCAUUGUUUCCUCAGCCGCCGUCAGCGUCGCUCCUACCGCCGCCGAGAAAGUCCCCGCGGAGUGCCCCGAACUAACGCGCCGAUGCUUGUUGGGUGAGGUGUUUGAGGGUGACAAGUAUGAAAGUUGGCUGCGCCCGUUGGUGAAUGUUACCGGGCGCGAUGGCCCGCUAUCGCAACUUAUCCGUUACCGUCCCGUUACGCCGGAGGCCGCCAACUCCGUGCUGUUGGACGAGGCUUUCCUGGACACUCUGGCCCUGCUGUACAACAAUCCGGAUCAAUUGCGGGCCCUGCUGACGCUGUUGAGCUCGGACACAGCGCCGCGCUGGAUGACGGUGAUGCGCGGCUACAGCGAGUGCGGCGAUGGCUCGCCGGCCGUGUACACGUGCGUGGACGACCUGUGCCGCGGCUACGACCUCACGCGACUGUCAUACGGGCGCAGCAUCUUCACGGAACACGUGUUAGGCUUCGAGCUGGUGCCACCGUCUCUCUUUAACGUGGUGGUGGCCAUACGCAACGAAGCCACGCGUACCAACCGCGCCGUGCGUCUGCCCGUGAGCACCGCUGCCGCGCCCGAGGGCAUCACGCUCUUUUACGGCCUGUACAACGCAGUGAAGGAAUUCUGCCUGCGUCACCAGCUGGACCCGCCGCUGCUACGCCACCUAGAUAAAUACUACGCCGGACUGCCGCCCGAGCUGAAGCAGACGCGCGUCAACCUGCCGGCUCACUCGCGCUAUGGCCCUCAAGCAGUGGAUGCUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV gL dimer(SEQ ID NO: 21)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGTGGCTCTTATATTTCTTCTTACTCTTCTTTTCTCTCTTATTTCCATGTGCCGCCGCCCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCCTCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAGGTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGTTACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCGGATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGCGATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTCACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAACCGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTCTGCCTGCGTCACCAGCTGGACCCGCCGCTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGCGTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV gL dimer(SEQ ID NO: 114)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGUGGCUCUUAUAUUUCUUCUUACUCUUCUUUUCUCUCUUAUUUCCAUGUGCCGCCGCCCGGAUUGCGGCUUCUCUUUCUCACCUGGACCGGUGAUACUGCUGUGGUGUUGCCUUCUGCUGCCCAUUGUUUCCUCAGCCGCCGUGAGCGUCGCUCCUACCGCCGCCGAGAAAGUCCCCGCGGAGUGCCCCGAACUAACGCGCCGAUGCUUGUUGGGUGAGGUGUUUGAGGGUGACAAGUAUGAAAGUUGGCUGCGCCCGUUGGUGAAUGUUACCGGGCGCGAUGGCCCGCUAUCGCAACUUAUCCGUUACCGUCCCGUUACGCCGGAGGCCGCCAACUCCGUGCUGUUGGACGAGGCUUUCCUGGACACUCUGGCCCUGCUGUACAACAAUCCGGAUCAAUUGCGGGCCCUGCUGACGCUGUUGAGCUCGGACACAGCGCCGCGCUGGAUGACGGUGAUGCGCGGCUACAGCGAGUGCGGCGAUGGCUCGCCGGCCGUGUACACGUGCGUGGACGACCUGUGCCGCGGCUACGACCUCACGCGACUGUCAUACGGGCGCAGCAUCUUCACGGAACACGUGUUAGGCUUCGAGCUGGUGCCACCGUCUCUCUUUAACGUGGUGGUGGCCAUACGCAACGAAGCCACGCGUACCAACCGCGCCGUGCGUCUGCCCGUGAGCACCGCUGCCGCGCCCGAGGGCAUCACGCUCUUUUACGGCCUGUACAACGCAGUGAAGGAAUUCUGCCUGCGUCACCAGCUGGACCCGCCGCUGCUACGCCACCUAGAUAAAUACUACGCCGGACUGCCGCCCGAGCUGAAGCAGACGCGCGUCAACCUGCCGGCUCACUCGCGCUAUGGCCCUCAAGCAGUGGAUGCUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV UL128 penta(SEQ ID NO: 22)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGTTCGGCTGGTACAGGCTAACCAGAAGACAGATAAGAGCCTCCATGAGTCCCAAAGATCTGACGCCGTTCTTGACGGCGTTGTGGCTGCTATTGGGTCACAGCCGCGTGCCGCGGGTGCGCGCAGAAGAATGTTGCGAATTCATAAACGTCAACCACCCGCCGGAACGCTGTTACGATTTCAAAATGTGCAATCGCTTCACCGTCGCGCTGCGGTGTCCGGACGGCGAAGTCTGCTACAGTCCCGAGAAAACGGCTGAGATTCGCGGGATCGTCACCACCATGACCCATTCATTGACACGCCAGGTCGTACACAACAAACTGACGAGCTGCAACTACAATCCGTTATACCTCGAAGCTGACGGGCGAATACGCTGCGGCAAAGTAAACGACAAGGCGCAGTACCTGCTGGGCGCCGCTGGCAGCGTTCCCTATCGATGGATCAATCTGGAATACGACAAGATAACCCGGATCGTGGGCCTGGATCAGTACCTGGAGAGCGTTAAGAAACACAAACGGCTGGATGTGTGCCGCGCTAAAATGGGCTATATGCTGCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV UL128 penta(SEQ ID NO: 99)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGUUCGGCUGGUACAGGCUAACCAGAAGACAGAUAAGAGCCUCCAUGAGUCCCAAAGAUCUGACGCCGUUCUUGACGGCGUUGUGGCUGCUAUUGGGUCACAGCCGCGUGCCGCGGGUGCGCGCAGAAGAAUGUUGCGAAUUCAUAAACGUCAACCACCCGCCGGAACGCUGUUACGAUUUCAAAAUGUGCAAUCGCUUCACCGUCGCGCUGCGGUGUCCGGACGGCGAAGUCUGCUACAGUCCCGAGAAAACGGCUGAGAUUCGCGGGAUCGUCACCACCAUGACCCAUUCAUUGACACGCCAGGUCGUACACAACAAACUGACGAGCUGCAACUACAAUCCGUUAUACCUCGAAGCUGACGGGCGAAUACGCUGCGGCAAAGUAAACGACAAGGCGCAGUACCUGCUGGGCGCCGCUGGCAGCGUUCCCUAUCGAUGGAUCAAUCUGGAAUACGACAAGAUAACCCGGAUCGUGGGCCUGGAUCAGUACCUGGAGAGCGUUAAGAAACACAAACGGCUGGAUGUGUGCCGCGCUAAAAUGGGCUAUAUGCUGCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-UL130 penta(SEQ ID NO: 23)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAGGCTCTTATCTGTCTTCTCAGTCCGAATTCGAAGTACGGCTACCATGCTGCGGCTTCTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCCTGTCTGGCGTCTCCGTGGTCGACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCTTTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACCCTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGTCGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAGATTTTTGGAGCGCACATGGTGCCCAAGCAGACCAAGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATGAAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACTTACACCTTCTGCACCCATCCCAATCTCATCGTTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UL130 penta(SEQ ID NO: 100)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAGGCUCUUAUCUGUCUUCUCAGUCCGAAUUCGAAGUACGGCUACCAUGCUGCGGCUUCUGCUUCGUCACCACUUUCACUGCCUGCUUCUGUGCGCGGUUUGGGCAACGCCCUGUCUGGCGUCUCCGUGGUCGACGCUAACAGCAAACCAGAAUCCGUCCCCGCCAUGGUCUAAACUGACGUAUUCCAAACCGCAUGACGCGGCGACGUUUUACUGUCCUUUUCUCUAUCCCUCGCCCCCACGAUCCCCCUUGCAAUUCUCGGGGUUCCAGCGGGUAUCAACGGGUCCCGAGUGUCGCAACGAGACCCUGUAUCUGCUGUACAACCGGGAAGGCCAGACCUUGGUGGAGAGAAGCUCCACCUGGGUGAAAAAGGUGAUCUGGUACCUGAGCGGUCGGAACCAAACCAUCCUCCAACGGAUGCCCCGAACGGCUUCGAAACCGAGCGACGGAAACGUGCAGAUCAGCGUGGAAGACGCCAAGAUUUUUGGAGCGCACAUGGUGCCCAAGCAGACCAAGCUGCUACGCUUCGUCGUCAACGAUGGCACACGUUAUCAGAUGUGUGUGAUGAAGCUGGAGAGCUGGGCUCACGUCUUCCGGGACUACAGCGUGUCUUUUCAGGUGCGAUUGACGUUCACCGAGGCCAAUAACCAGACUUACACCUUCUGCACCCAUCCCAAUCUCAUCGUUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMVUL130 trimer(SEQ ID NO: 24)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGTGGCTCTTATATTTCTTCTTAGTCCGAATTCGAAGTACGGCTACATGCTGCGGCTTCTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCGTGTCTGGCGTCTCCGTGGTCGACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCTTTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACCCTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGTCGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAGATTTTTGGAGCGCACATGGTGCCCAAGCAGACCAAGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATGAAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACTTACACCTTCTGCACCCATCCCAATCTCATCGTTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMVUL130 trimer(SEQ ID NO: 115)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGUGGCUCUUAUAUUUCUUCUUAGUCCGAAUUCGAAGUACGGCUACAUGCUGCGGCUUCUGCUUCGUCACCACUUUCACUGCCUGCUUCUGUGCGCGGUUUGGGCAACGCCCUGUCUGGCGUCUCCGUGGUCGACGCUAACAGCAAACCAGAAUCCGUCCCCGCCAUGGUCUAAACUGACGUAUUCCAAACCGCAUGACGCGGCGACGUUUUACUGUCCUUUUCUCUAUCCCUCGCCCCCACGAUCCCCCUUGCAAUUCUCGGGGUUCCAGCGGGUAUCAACGGGUCCCGAGUGUCGCAACGAGACCCUGUAUCUGCUGUACAACCGGGAAGGCCAGACCUUGGUGGAGAGAAGCUCCACCUGGGUGAAAAAGGUGAUCUGGUACCUGAGCGGUCGGAACCAAACCAUCCUCCAACGGAUGCCCCGAACGGCUUCGAAACCGAGCGACGGAAACGUGCAGAUCAGCGUGGAAGACGCCAAGAUUUUUGGAGCGCACAUGGUGCCCAAGCAGACCAAGCUGCUACGCUUCGUCGUCAACGAUGGCACACGUUAUCAGAUGUGUGUGAUGAAGCUGGAGAGCUGGGCUCACGUCUUCCGGGACUACAGCGUGUCUUUUCAGGUGCGAUUGACGUUCACCGAGGCCAAUAACCAGACUUACACCUUCUGCACCCAUCCCAAUCUCAUCGUUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-UL131A penta(SEQ ID NO: 25)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGTAGCCGTACTTCGAATTCGGACAAGCTTCTCTCTCGTCTGTCCATGCGGCTGTGTCGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGGAAACCGCGGAAAAAAACGATTATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGGCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGACCTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGAATCAACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCCGCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UL131A penta(SEQ ID NO: 101)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGUAGCCGUACUUCGAAUUCGGACAAGCUUCUCUCUCGUCUGUCCAUGCGGCUGUGUCGGGUGUGGCUGUCUGUUUGUCUGUGCGCCGUGGUGCUGGGUCAGUGCCAGCGGGAAACCGCGGAAAAAAACGAUUAUUACCGAGUACCGCAUUACUGGGACGCGUGCUCUCGCGCGCUGCCCGACCAAACCCGUUACAAGUAUGUGGAACAGCUCGUGGACCUCACGUUGAACUACCACUACGAUGCGAGCCACGGCUUGGACAACUUUGACGUGCUCAAGAGAAUCAACGUGACCGAGGUGUCGUUGCUCAUCAGCGACUUUAGACGUCAGAACCGUCGCGGCGGCACCAACAAAAGGACCACGUUCAACGCCGCCGGUUCGCUGGCGCCACACGCCCGGAGCCUCGAGUUCAGCGUGCGGCUCUUUGCCAACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMVUL131A trimer(SEQ ID NO: 26)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGTAGCCGTACTTCGAATTCGGACTTTCTTTTCTCTCTTATTTCCATGCGGCTGTGTCGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGGAAACCGCGGAAAAAAACGATTATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGCCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGACCTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGAATCAACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCCGCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMVUL131A trimer(SEQ ID NO: 102)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGUAGCCGUACUUCGAAUUCGGACUUUCUUUUCUCUCUUAUUUCCAUGCGGCUGUGUCGGGUGUGGCUGUCUGUUUGUCUGUGCGCCGUGGUGCUGGGUCAGUGCCAGCGGGAAACCGCGGAAAAAAACGAUUAUUACCGAGUACCGCAUUACUGGGACGCGUGCUCUCGCGCGCUGCCCGACCAAACCCGUUACAAGUAUGUGGAACAGCUCGUGGACCUCACGUUGAACUACCACUACGAUGCGAGCCACGGCUUGGACAACUUUGACGUGCUCAAGAGAAUCAACGUGACCGAGGUGUCGUUGCUCAUCAGCGACUUUAGACGUCAGAACCGUCGCGGCGGCACCAACAAAAGGACCACGUUCAACGCCGCCGGUUCGCUGGCGCCACACGCCCGGAGCCUCGAGUUCAGCGUGCGGCUCUUUGCCAACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Example 16: hCMV Vaccine—hCMV UL Fusion Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

hCMV pp65-IE1, hCMV UL83-UL123 fusion (SEQ ID NO: 27)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTCGCGCGGTCGCCGTTGTCCCGAAATGATATCCGTACTGGGTCCCATTTCGGGGCACGTGCTGAAAGCCGTGTTTAGTCGCGGCGATACGCCGGTGCTGCCGCACGAGACGCGACTCCTGCAGACGGGTATCCACGTACGCGTGAGCCAGCCCTCGCTGATCCTGGTGTCGCAGTACACGCCCGACTCGACGCCATGCCACCGCGGCGACAATCAGCTGCAGGTGCAGCACACGTACTTTACGGGCAGCGAGGTGGAGAACGTGTCGGTCAACGTGCACAACCCCACGGGCCGAAGCATCTGCCCCAGCCAAGAGCCCATGTCGATCTATGTGTACGCGCTGCCGCTCAAGATGCTGAACATCCCCAGCATCAACGTGCACCACTACCCGTCGGCGGCCGAGCGCAAACACCGACACCTGCCCGTAGCCGACGCTGTTATTCACGCGTCGGGCAAGCAGATGTGGCAGGCGCGTCTCACGGTCTCGGGACTGGCCTGGACGCGTCAGCAGAACCAGTGGAAAGAGCCCGACGTCTACTACACGTCAGCGTTCGTGTTTCCCACCAAGGACGTGGCACTGCGGCACGTGGTGTGCGCGCACGAGCTGGTTTGCTCCATGGAGAACACGCGCGCAACCAAGATGCAGGTGATAGGTGACCAGTACGTCAAGGTGTACCTGGAGTCCTTCTGCGAGGACGTGCCCTCCGGCAAGCTCTTTATGCACGTCACGCTGGGCTCTGACGTGGAAGAGGACCTAACGATGACCCGCAACCCGCAACCCTTCATGCGCCCCCACGAGCGCAACGGCTTTACGGTGTTGTGTCCCAAAAATATGATAATCAAACCGGGCAAGATCTCGCACATCATGCTGGATGTGGCTTTTACCTCACACGAGCATTTTGGGCTGCTGTGTCCCAAGAGCATCCCGGGCCTGAGCATCTCAGGTAACCTGTTGATGAACGGGCAGCAAATCTTCCTGGAGGTACAAGCGATACGCGAGACCGTGGAACTGCGTCAGTACGATCCCGTGGCTGCGCTCTTCTTTTTCGATATCGACTTGTTGCTGCAGCGCGGGCCTCAGTACAGCGAGCACCCCACCTTCACCAGCCAGTATCGCATCCAGGGCAAGCTTGAGTACCGACACACCTGGGACCGGCACGACGAGGGTGCCGCCCAGGGCGACGACGACGTCTGGACCAGCGGATCGGACTCCGACGAAGAACTCGTAACCACCGAGCGTAAGACGCCCCGCGTCACCGGCGGCGGCGCCATGGCGAGCGCCTCCACTTCCGCGGGCCGCAAACGCAAATCAGCATCCTCGGCGACGGCGTGCACGGCGGGCGTTATGACACGCGGCCGCCTTAAGGCCGAGTCCACCGTCGCGCCCGAAGAGGACACCGACGAGGATTCCGACAACGAAATCCACAATCCGGCCGTGTTCACCTGGCCGCCCTGGCAGGCCGGCATCCTGGCCCGCAACCTGGTGCCCATGGTGGCTACGGTTCAGGGTCAGAATCTGAAGTACCAGGAGTTCTTCTGGGACGCCAACGACATCTACCGCATCTTCGCCGAATTGGAAGGCGTATGGCAGCCCGCTGCGCAACCCAAACGTCGCCGCCACCGGCAAGACGCCTTGCCCGGGCCATGCATCGCCTCGACGCCCAAAAAGCACCGAGGTGAGTCCTCTGCCAAGAGAAAGATGGACCCTGATAATCCTGACGAGGGCCCTTCCTCCAAGGTGCCACGGCCCGAGACACCCGTGACCAAGGCCACGACGTTCCTGCAGACTATGTTAAGGAAGGAGGTTAACAGTCAGCTGAGCCTGGGAGACCCGCTGTTCCCAGAATTGGCCGAAGAATCCCTCAAAACCTTTGAACAAGTGACCGAGGATTGCAACGAGAACCCCGAAAAAGATGTCCTGACAGAACTCGTCAAACAGATTAAGGTTCGAGTGGACATGGTGCGGCATAGAATCAAGGAGCACATGCTGAAAAAATATACCCAGACGGAAGAAAAATTCACTGGCGCCTTTAATATGATGGGAGGATGTTTGCAGAATGCCTTAGATATCTTAGATAAGGTTCATGAGCCTTTCGAGGACATGAAGTGTATTGGGCTAACTATGCAGAGCATGTATGAGAACTACATTGTACCTGAGGATAAGCGGGAGATGTGGATGGCTTGTATTAAGGAGCTGCATGATGTGAGCAAGGGCGCCGCTAACAAGTTGGGGGGTGCACTGCAGGCTAAGGCCCGTGCTAAAAAGGATGAACTTAGGAGAAAGATGATGTATATGTGCTACAGGAATATAGAGTTCTTTACCAAGAACTCAGCCTTCCCTAAGACCACCAATGGCTGCAGTCAGGCCATGGCGGCATTGCAGAACTTGCCTCAGTGCTCTCCTGATGAGATTATGTCTTATGCCCAGAAAATCTTTAAGATTTTGGATGAGGAGAGAGACAAGGTGCTCACGCACATTGATCACATATTTATGGATATCCTCACTACATGTGTGGAAACAATGTGTAATGAGTACAAGGTCACTAGTGACGCTTGTATGATGACCATGTACGGGGGCATCTCTCTCTTAAGTGAGTTCTGTCGGGTGCTGTGCTGCTATGTCTTAGAGGAGACTAGTGTGATGCTGGCCAAGCGGCCTCTGATAACCAAGCCTGAGGTTATCAGTGTAATGAAGCGCCGCATTGAGGAGATCTGCATGAAGGTCTTTGCCCAGTACATTCTGGGGGCCGATCCTTTGAGAGTCTGCTCTCCTAGTGTGGATGACCTACGGGCCATCGCCGAGGAGTCAGATGAGGAAGAGGCTATTGTAGCCTACACTTTGGCCACCGCTGGTGCCAGCTCCTCTGATTCTCTGGTGTCACCTCCAGAGTCCCCTGTACCCGCGACTATCCCTCTGTCCTCAGTAATTGTGGCTGAGAACAGTGATCAGGAAGAAAGTGAACAGAGTGATGAGGAACAGGAGGAGGGTGCTCAGGAGGAGCGGGAGGACACTGTGTCTGTCAAGTCTGAGCCAGTGTCTGAGATAGAGGAAGTTGCCTCAGAGGAAGAGGAGGATGGTGCTGAGGAACCCACCGCCTCTGGAGGCAAGAGCACCCACCCTATGGTGACTAGAAGCAAGGCTGACCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV pp65-IE1, hCMV UL83-UL123 fusion (SEQ ID NO: 103)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGUCGCGCGGUCGCCGUUGUCCCGAAAUGAUAUCCGUACUGGGUCCCAUUUCGGGGCACGUGCUGAAAGCCGUGUUUAGUCGCGGCGAUACGCCGGUGCUGCCGCACGAGACGCGACUCCUGCAGACGGGUAUCCACGUACGCGUGAGCCAGCCCUCGCUGAUCCUGGUGUCGCAGUACACGCCCGACUCGACGCCAUGCCACCGCGGCGACAAUCAGCUGCAGGUGCAGCACACGUACUUUACGGGCAGCGAGGUGGAGAACGUGUCGGUCAACGUGCACAACCCCACGGGCCGAAGCAUCUGCCCCAGCCAAGAGCCCAUGUCGAUCUAUGUGUACGCGCUGCCGCUCAAGAUGCUGAACAUCCCCAGCAUCAACGUGCACCACUACCCGUCGGCGGCCGAGCGCAAACACCGACACCUGCCCGUAGCCGACGCUGUUAUUCACGCGUCGGGCAAGCAGAUGUGGCAGGCGCGUCUCACGGUCUCGGGACUGGCCUGGACGCGUCAGCAGAACCAGUGGAAAGAGCCCGACGUCUACUACACGUCAGCGUUCGUGUUUCCCACCAAGGACGUGGCACUGCGGCACGUGGUGUGCGCGCACGAGCUGGUUUGCUCCAUGGAGAACACGCGCGCAACCAAGAUGCAGGUGAUAGGUGACCAGUACGUCAAGGUGUACCUGGAGUCCUUCUGCGAGGACGUGCCCUCCGGCAAGCUCUUUAUGCACGUCACGCUGGGCUCUGACGUGGAAGAGGACCUAACGAUGACCCGCAACCCGCAACCCUUCAUGCGCCCCCACGAGCGCAACGGCUUUACGGUGUUGUGUCCCAAAAAUAUGAUAAUCAAACCGGGCAAGAUCUCGCACAUCAUGCUGGAUGUGGCUUUUACCUCACACGAGCAUUUUGGGCUGCUGUGUCCCAAGAGCAUCCCGGGCCUGAGCAUCUCAGGUAACCUGUUGAUGAACGGGCAGCAAAUCUUCCUGGAGGUACAAGCGAUACGCGAGACCGUGGAACUGCGUCAGUACGAUCCCGUGGCUGCGCUCUUCUUUUUCGAUAUCGACUUGUUGCUGCAGCGCGGGCCUCAGUACAGCGAGCACCCCACCUUCACCAGCCAGUAUCGCAUCCAGGGCAAGCUUGAGUACCGACACACCUGGGACCGGCACGACGAGGGUGCCGCCCAGGGCGACGACGACGUCUGGACCAGCGGAUCGGACUCCGACGAAGAACUCGUAACCACCGAGCGUAAGACGCCCCGCGUCACCGGCGGCGGCGCCAUGGCGAGCGCCUCCACUUCCGCGGGCCGCAAACGCAAAUCAGCAUCCUCGGCGACGGCGUGCACGGCGGGCGUUAUGACACGCGGCCGCCUUAAGGCCGAGUCCACCGUCGCGCCCGAAGAGGACACCGACGAGGAUUCCGACAACGAAAUCCACAAUCCGGCCGUGUUCACCUGGCCGCCCUGGCAGGCCGGCAUCCUGGCCCGCAACCUGGUGCCCAUGGUGGCUACGGUUCAGGGUCAGAAUCUGAAGUACCAGGAGUUCUUCUGGGACGCCAACGACAUCUACCGCAUCUUCGCCGAAUUGGAAGGCGUAUGGCAGCCCGCUGCGCAACCCAAACGUCGCCGCCACCGGCAAGACGCCUUGCCCGGGCCAUGCAUCGCCUCGACGCCCAAAAAGCACCGAGGUGAGUCCUCUGCCAAGAGAAAGAUGGACCCUGAUAAUCCUGACGAGGGCCCUUCCUCCAAGGUGCCACGGCCCGAGACACCCGUGACCAAGGCCACGACGUUCCUGCAGACUAUGUUAAGGAAGGAGGUUAACAGUCAGCUGAGCCUGGGAGACCCGCUGUUCCCAGAAUUGGCCGAAGAAUCCCUCAAAACCUUUGAACAAGUGACCGAGGAUUGCAACGAGAACCCCGAAAAAGAUGUCCUGACAGAACUCGUCAAACAGAUUAAGGUUCGAGUGGACAUGGUGCGGCAUAGAAUCAAGGAGCACAUGCUGAAAAAAUAUACCCAGACGGAAGAAAAAUUCACUGGCGCCUUUAAUAUGAUGGGAGGAUGUUUGCAGAAUGCCUUAGAUAUCUUAGAUAAGGUUCAUGAGCCUUUCGAGGACAUGAAGUGUAUUGGGCUAACUAUGCAGAGCAUGUAUGAGAACUACAUUGUACCUGAGGAUAAGCGGGAGAUGUGGAUGGCUUGUAUUAAGGAGCUGCAUGAUGUGAGCAAGGGCGCCGCUAACAAGUUGGGGGGUGCACUGCAGGCUAAGGCCCGUGCUAAAAAGGAUGAACUUAGGAGAAAGAUGAUGUAUAUGUGCUACAGGAAUAUAGAGUUCUUUACCAAGAACUCAGCCUUCCCUAAGACCACCAAUGGCUGCAGUCAGGCCAUGGCGGCAUUGCAGAACUUGCCUCAGUGCUCUCCUGAUGAGAUUAUGUCUUAUGCCCAGAAAAUCUUUAAGAUUUUGGAUGAGGAGAGAGACAAGGUGCUCACGCACAUUGAUCACAUAUUUAUGGAUAUCCUCACUACAUGUGUGGAAACAAUGUGUAAUGAGUACAAGGUCACUAGUGACGCUUGUAUGAUGACCAUGUACGGGGGCAUCUCUCUCUUAAGUGAGUUCUGUCGGGUGCUGUGCUGCUAUGUCUUAGAGGAGACUAGUGUGAUGCUGGCCAAGCGGCCUCUGAUAACCAAGCCUGAGGUUAUCAGUGUAAUGAAGCGCCGCAUUGAGGAGAUCUGCAUGAAGGUCUUUGCCCAGUACAUUCUGGGGGCCGAUCCUUUGAGAGUCUGCUCUCCUAGUGUGGAUGACCUACGGGCCAUCGCCGAGGAGUCAGAUGAGGAAGAGGCUAUUGUAGCCUACACUUUGGCCACCGCUGGUGCCAGCUCCUCUGAUUCUCUGGUGUCACCUCCAGAGUCCCCUGUACCCGCGACUAUCCCUCUGUCCUCAGUAAUUGUGGCUGAGAACAGUGAUCAGGAAGAAAGUGAACAGAGUGAUGAGGAACAGGAGGAGGGUGCUCAGGAGGAGCGGGAGGACACUGUGUCUGUCAAGUCUGAGCCAGUGUCUGAGAUAGAGGAAGUUGCCUCAGAGGAAGAGGAGGAUGGUGCUGAGGAACCCACCGCCUCUGGAGGCAAGAGCACCCACCCUAUGGUGACUAGAAGCAAGGCUGACCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV pp65-IE1FLAG, hCMV UL83-UL123 FLAG tag(SEQ ID NO: 28)TCAAGCTTTTGGACCCTCGTAGAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTCGCGCGGTCGCCGTTGTCCCGAAATGATATCCGTACTGGGTCCCATTTCGGGGCACGTGCTGAAAGCCGTGTTTAGTCGCGGCGATACGCCGGTGCTGCCGCACGAGACGCGACTCCTGCAGACGGGTATCCACGTACGCGTGAGCCAGCCCTCGCTGATCCTGGTGTCGCAGTACACGCCCGACTCGACGCCATGCCACCGCGGCGACAATCAGCTGCAGGTGCAGCACACGTACTTTACGGGCAGCGAGGTGGAGAACGTGTCGGTCAACGTGCACAACCCCACGGGCCGAAGCATCTGCCCCAGCCAAGAGCCCATGTCGATCTATGTGTACGCGCTGCCGCTCAAGATGCTGAACATCCCCAGCATCAACGTGCACCACTACCCGTCGGCGGCCGAGCGCAAACACCGACACCTGCCCGTAGCCGACGCTGTTATTCACGCGTCGGGCAAGCAGATGTGGCAGGCGCGTCTCACGGTCTCGGGACTGGCCTGGACGCGTCAGCAGAACCAGTGGAAAGAGCCCGACGTCTACTACACGTCAGCGTTCGTGTTTCCCACCAAGGACGTGGCACTGCGGCACGTGGTGTGCGCGCACGAGCTGGTTTGCTCCATGGAGAACACGCGCGCAACCAAGATGCAGGTGATAGGTGACCAGTACGTCAAGGTGTACCTGGAGTCCTTCTGCGAGGACGTGCCCTCCGGCAAGCTCTTTATGCACGTCACGCTGGGCTCTGACGTGGAAGAGGACCTAACGATGACCCGCAACCCGCAACCCTTCATGCGCCCCCACGAGCGCAACGGCTTTACGGTGTTGTGTCCCAAAAATATGATAATCAAACCGGGCAAGATCTCGCACATCATGCTGGATGTGGCTTTTACCTCACACGAGCATTTTGGGCTGCTGTGTCCCAAGAGCATCCCGGGCCTGAGCATCTCAGGTAACCTGTTGATGAACGGGCAGCAAATCTTCCTGGAGGTACAAGCGATACGCGAGACCGTGGAACTGCGTCAGTACGATCCCGTGGCTGCGCTCTTCTTTTTCGATATCGACTTGTTGCTGCAGCGCGGGCCTCAGTACAGCGAGCACCCCACCTTCACCAGCCAGTATCGCATCCAGGGCAAGCTTGAGTACCGACACACCTGGGACCGGCACGACGAGGGTGCCGCCCAGGGCGACGACGACGTCTGGACCAGCGGATCGGACTCCGACGAAGAACTCGTAACCACCGAGCGTAAGACGCCCCGCGTCACCGGCGGCGGCGCCATGGCGAGCGCCTCCACTTCCGCGGGCCGCAAACGCAAATCAGCATCCTCGGCGACGGCGTGCACGGCGGGCGTTATGACACGCGGCCGCCTTAAGGCCGAGTCCACCGTCGCGCCCGAAGAGGACACCGACGAGGATTCCGACAACGAAATCCACAATCCGGCCGTGTTCACCTGGCCGCCCTGGCAGGCCGGCATCCTGGCCCGCAACCTGGTGCCCATGGTGGCTACGGTTCAGGGTCAGAATCTGAAGTACCAGGAGTTCTTCTGGGACGCCAACGACATCTACCGCATCTTCGCCGAATTGGAAGGCGTATGGCAGCCCGCTGCGCAACCCAAACGTCGCCGCCACCGGCAAGACGCCTTGCCCGGGCCATGCATCGCCTCGACGCCCAAAAAGCACCGAGGTGAGTCCTCTGCCAAGAGAAAGATGGACCCTGATAATCCTGACGAGGGCCCTTCCTCCAAGGTGCCACGGCCCGAGACACCCGTGACCAAGGCCACGACGTTCCTGCAGACTATGTTAAGGAAGGAGGTTAACAGTCAGCTGAGCCTGGGAGACCCGCTGTTCCCAGAATTGGCCGAAGAATCCCTCAAAACCTTTGAACAAGTGACCGAGGATTGCAACGAGAACCCCGAAAAAGATGTCCTGACAGAACTCGTCAAACAGATTAAGGTTCGAGTGGACATGGTGCGGCATAGAATCAAGGAGCACATGCTGAAAAAATATACCCAGACGGAAGAAAAATTCACTGGCGCCTTTAATATGATGGGAGGATGTTTGCAGAATGCCTTAGATATCTTAGATAAGGTTCATGAGCCTTTCGAGGACATGAAGTGTATTGGGCTAACTATGCAGAGCATGTATGAGAACTACATTGTACCTGAGGATAAGCGGGAGATGTGGATGGCTTGTATTAAGGAGCTGCATGATGTGAGCAAGGGCGCCGCTAACAAGTTGGGGGGTGCACTGCAGGCTAAGGCCCGTGCTAAAAAGGATGAACTTAGGAGAAAGATGATGTATATGTGCTACAGGAATATAGAGTTCTTTACCAAGAACTCAGCCTTCCCTAAGACCACCAATGGCTGCAGTCAGGCCATGGCGGCATTGCAGAACTTGCCTCAGTGCTCTCCTGATGAGATTATGTCTTATGCCCAGAAAATCTTTAAGATTTTGGATGAGGAGAGAGACAAGGTGCTCACGCACATTGATCACATATTTATGGATATCCTCACTACATGTGTGGAAACAATGTGTAATGAGTACAAGGTCACTAGTGACGCTTGTATGATGACCATGTACGGGGGCATCTCTCTCTTAAGTGAGTTCTGTCGGGTGCTGTGCTGCTATGTCTTAGAGGAGACTAGTGTGATGCTGGCCAAGCGGCCTCTGATAACCAAGCCTGAGGTTATCAGTGTAATGAAGCGCCGCATTGAGGAGATCTGCATGAAGGTCTTTGCCCAGTACATTCTGGGGGCCGATCCTTTGAGAGTCTGCTCTCCTAGTGTGGATGACCTACGGGCCATCGCCGAGGAGTCAGATGAGGAAGAGGCTATTGTAGCCTACACTTTGGCCACCGCTGGTGCCAGCTCCTCTGATTCTCTGGTGTCACCTCCAGAGTCCCCTGTACCCGCGACTATCCCTCTGTCCTCAGTAATTGTGGCTGAGAACAGTGATCAGGAAGAAAGTGAACAGAGTGATGAGGAACAGGAGGAGGGTGCTCAGGAGGAGCGGGAGGACACTGTGTCTGTCAAGTCTGAGCCAGTGTCTGAGATAGAGGAAGTTGCCTCAGAGGAAGAGGAGGATGGTGCTGAGGAACCCACCGCCTCTGGAGGCAAGAGCACCCACCCTATGGTGACTAGAAGCAAGGCTGACCAGGATTACAAGGACGATGACGATAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGChCMV pp65-IE1, hCMV UL83-UL123 fusion (SEQ ID NO: 104)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGUCGCGCGGUCGCCGUUGUCCCGAAAUGAUAUCCGUACUGGGUCCCAUUUCGGGGCACGUGCUGAAAGCCGUGUUUAGUCGCGGCGAUACGCCGGUGCUGCCGCACGAGACGCGACUCCUGCAGACGGGUAUCCACGUACGCGUGAGCCAGCCCUCGCUGAUCCUGGUGUCGCAGUACACGCCCGACUCGACGCCAUGCCACCGCGGCGACAAUCAGCUGCAGGUGCAGCACACGUACUUUACGGGCAGCGAGGUGGAGAACGUGUCGGUCAACGUGCACAACCCCACGGGCCGAAGCAUCUGCCCCAGCCAAGAGCCCAUGUCGAUCUAUGUGUACGCGCUGCCGCUCAAGAUGCUGAACAUCCCCAGCAUCAACGUGCACCACUACCCGUCGGCGGCCGAGCGCAAACACCGACACCUGCCCGUAGCCGACGCUGUUAUUCACGCGUCGGGCAAGCAGAUGUGGCAGGCGCGUCUCACGGUCUCGGGACUGGCCUGGACGCGUCAGCAGAACCAGUGGAAAGAGCCCGACGUCUACUACACGUCAGCGUUCGUGUUUCCCACCAAGGACGUGGCACUGCGGCACGUGGUGUGCGCGCACGAGCUGGUUUGCUCCAUGGAGAACACGCGCGCAACCAAGAUGCAGGUGAUAGGUGACCAGUACGUCAAGGUGUACCUGGAGUCCUUCUGCGAGGACGUGCCCUCCGGCAAGCUCUUUAUGCACGUCACGCUGGGCUCUGACGUGGAAGAGGACCUAACGAUGACCCGCAACCCGCAACCCUUCAUGCGCCCCCACGAGCGCAACGGCUUUACGGUGUUGUGUCCCAAAAAUAUGAUAAUCAAACCGGGCAAGAUCUCGCACAUCAUGCUGGAUGUGGCUUUUACCUCACACGAGCAUUUUGGGCUGCUGUGUCCCAAGAGCAUCCCGGGCCUGAGCAUCUCAGGUAACCUGUUGAUGAACGGGCAGCAAAUCUUCCUGGAGGUACAAGCGAUACGCGAGACCGUGGAACUGCGUCAGUACGAUCCCGUGGCUGCGCUCUUCUUUUUCGAUAUCGACUUGUUGCUGCAGCGCGGGCCUCAGUACAGCGAGCACCCCACCUUCACCAGCCAGUAUCGCAUCCAGGGCAAGCUUGAGUACCGACACACCUGGGACCGGCACGACGAGGGUGCCGCCCAGGGCGACGACGACGUCUGGACCAGCGGAUCGGACUCCGACGAAGAACUCGUAACCACCGAGCGUAAGACGCCCCGCGUCACCGGCGGCGGCGCCAUGGCGAGCGCCUCCACUUCCGCGGGCCGCAAACGCAAAUCAGCAUCCUCGGCGACGGCGUGCACGGCGGGCGUUAUGACACGCGGCCGCCUUAAGGCCGAGUCCACCGUCGCGCCCGAAGAGGACACCGACGAGGAUUCCGACAACGAAAUCCACAAUCCGGCCGUGUUCACCUGGCCGCCCUGGCAGGCCGGCAUCCUGGCCCGCAACCUGGUGCCCAUGGUGGCUACGGUUCAGGGUCAGAAUCUGAAGUACCAGGAGUUCUUCUGGGACGCCAACGACAUCUACCGCAUCUUCGCCGAAUUGGAAGGCGUAUGGCAGCCCGCUGCGCAACCCAAACGUCGCCGCCACCGGCAAGACGCCUUGCCCGGGCCAUGCAUCGCCUCGACGCCCAAAAAGCACCGAGGUGAGUCCUCUGCCAAGAGAAAGAUGGACCCUGAUAAUCCUGACGAGGGCCCUUCCUCCAAGGUGCCACGGCCCGAGACACCCGUGACCAAGGCCACGACGUUCCUGCAGACUAUGUUAAGGAAGGAGGUUAACAGUCAGCUGAGCCUGGGAGACCCGCUGUUCCCAGAAUUGGCCGAAGAAUCCCUCAAAACCUUUGAACAAGUGACCGAGGAUUGCAACGAGAACCCCGAAAAAGAUGUCCUGACAGAACUCGUCAAACAGAUUAAGGUUCGAGUGGACAUGGUGCGGCAUAGAAUCAAGGAGCACAUGCUGAAAAAAUAUACCCAGACGGAAGAAAAAUUCACUGGCGCCUUUAAUAUGAUGGGAGGAUGUUUGCAGAAUGCCUUAGAUAUCUUAGAUAAGGUUCAUGAGCCUUUCGAGGACAUGAAGUGUAUUGGGCUAACUAUGCAGAGCAUGUAUGAGAACUACAUUGUACCUGAGGAUAAGCGGGAGAUGUGGAUGGCUUGUAUUAAGGAGCUGCAUGAUGUGAGCAAGGGCGCCGCUAACAAGUUGGGGGGUGCACUGCAGGCUAAGGCCCGUGCUAAAAAGGAUGAACUUAGGAGAAAGAUGAUGUAUAUGUGCUACAGGAAUAUAGAGUUCUUUACCAAGAACUCAGCCUUCCCUAAGACCACCAAUGGCUGCAGUCAGGCCAUGGCGGCAUUGCAGAACUUGCCUCAGUGCUCUCCUGAUGAGAUUAUGUCUUAUGCCCAGAAAAUCUUUAAGAUUUUGGAUGAGGAGAGAGACAAGGUGCUCACGCACAUUGAUCACAUAUUUAUGGAUAUCCUCACUACAUGUGUGGAAACAAUGUGUAAUGAGUACAAGGUCACUAGUGACGCUUGUAUGAUGACCAUGUACGGGGGCAUCUCUCUCUUAAGUGAGUUCUGUCGGGUGCUGUGCUGCUAUGUCUUAGAGGAGACUAGUGUGAUGCUGGCCAAGCGGCCUCUGAUAACCAAGCCUGAGGUUAUCAGUGUAAUGAAGCGCCGCAUUGAGGAGAUCUGCAUGAAGGUCUUUGCCCAGUACAUUCUGGGGGCCGAUCCUUUGAGAGUCUGCUCUCCUAGUGUGGAUGACCUACGGGCCAUCGCCGAGGAGUCAGAUGAGGAAGAGGCUAUUGUAGCCUACACUUUGGCCACCGCUGGUGCCAGCUCCUCUGAUUCUCUGGUGUCACCUCCAGAGUCCCCUGUACCCGCGACUAUCCCUCUGUCCUCAGUAAUUGUGGCUGAGAACAGUGAUCAGGAAGAAAGUGAACAGAGUGAUGAGGAACAGGAGGAGGGUGCUCAGGAGGAGCGGGAGGACACUGUGUCUGUCAAGUCUGAGCCAGUGUCUGAGAUAGAGGAAGUUGCCUCAGAGGAAGAGGAGGAUGGUGCUGAGGAACCCACCGCCUCUGGAGGCAAGAGCACCCACCCUAUGGUGACUAGAAGCAAGGCUGACCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Example 17: hCMV Vaccine—hCMV Concatameric Sequences

A hCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences. In someembodiments, a hCMV vaccine may comprise at least one RNA polynucleotidecomprising at least one of the mRNA sequences listed below or at leastone fragment of the mRNA sequences listed below.

Example 18: Immunogenicity Study

The instant study is designed to test the immunogenicity in mice ofcandidate CMV vaccines comprising an mRNA polynucleotide encoding the gHand gL glycoproteins or the UL128, UL130, and UL131A polypeptidesobtained from MCMV.

Mice are vaccinated on week 0 and 4 via intramuscular (IM) orintradermal (ID) routes. One group remains unvaccinated and one isadministered inactivated MCMV. Serum is collected from each mouse onweeks 1, 3 (pre-dose) and 5. Individual bleeds are tested for anti-gHand anti-gL activity or anti-UL128, anti-UL130, and anti-UL131A viaELISA assay from all three time points, and pooled samples from week 5only are tested by Western blot using inactivated MCMV.

ELISA Immunoassays

Antibody production is measured in a sample by ELISA. Appropriatelydiluted samples were placed in 96-well plates precoated with a captureantibody directed against an epitope of the antibody. Serum samplestypically were diluted 1:100 for the assay. Incubation and washingprotocols were performed using routine methods. Data is read at 450 nmwith wavelength. Data is reported and plotted.

Example 19: MCMV Challenge

The instant study is designed to test the efficacy in mice of candidateCMV vaccines against a lethal challenge using a mouse CMV vaccinecomprising mRNAs encoding gH and gL or UL128, UL130, and UL131A. Due tothe strict species specificity of CMV infection, there is no animalmodel available for study of HCMV infection and immunity. Murinecytomegalovirus (MCMV) infection is the most widely used mouse modelsimulating HCMV infection. In the current study, the immunogenicity andprotective efficacy of MCMV gH, gL, UL128, UL130, UL131A antigens areinvestigated.

BALB/c mice are randomly divided into groups. The groups arerespectively immunized with (1) 10 μg gB (positive control), (2) 10 μggH and gL mRNAs (combination of separate sequences), (3) 10 μg gH-gLconcatamer mRNA (single sequence), (4) 10 μg UL128, UL130, UL131A mRNAs(combination of separate sequences), (5) 10 μg UL128-UL130-UL131Aconcatamer mRNA (single sequence), (6) 10 μg gH-gL-UL128-UL130-UL131Aconcatamer mRNA (single sequence) and (7) PBS. Mice are immunized twotimes (second dose at day 28) by injection into the right quadricepsmuscle (IM) or by intradermal administration (ID), and are challengedwith a lethal dose (5×LD50, 200 μl/mouse) of SG-MCMV (Smith strain, 10⁵PFU) by intraperitoneal injection. This infection causes systemic virusreplication in mice and death of all unvaccinated mice within one weekafter the challenge.

Endpoint is day 5 post infection, death or euthanasia. Animalsdisplaying severe illness as determined by >30% weight loss, extremelethargy or paralysis are euthanized. The protective effects of the DNAvaccines are evaluated comprehensively using infection symptoms of bodytemperature, weight loss, and survival. The mice are weighed andassessed daily in order to monitor weight loss, apparent physicalcondition (bristled hair and wounded skin), body temperature, andbehaviour. The mice are humanely euthanized via cervical dislocationafter chloroform (inhalation excess) in all cases in order to minimizeor avoid animal suffering.

Example 20: MCMV Neutralization Assay

Mice are immunized according to the methods in Example 18. Mouse serumsamples are collected 3 weeks after the second immunization. Serumsamples are stored at −20° C. until use. Neutralizing antibody directedagainst MCMV are determined by a plaque reduction assay, for example, asdescribed in Geoffroy F, et al., Murine cytomegalovirus inactivated bysodium periodate is innocuous and immunogenic in mice and protects themagainst death and infection. Vaccine. 1996; 14: 1686-1694.Decomplemented sera (30 μl) are serially diluted 2-fold with MEM. Eachdilution is mixed with 100 PFU MCMV in 30 μl of MEM and then incubated 1hour at 4° C. and 1 hour at 37° C. The mixture is layered onto 3T3monolayers and PFU are calculated by the standard plaque assay. Aneutralization titer is expressed as the highest serum dilution requiredto achieve a 50% reduction in the number of plaques.

Example 21: IFN-γ ELISPOT Assay

Mice are immunized with gH or gL, or co-immunized with gH/gL mRNAs twice(day 0 and day 28) at a dosage of 10 μg by IM. Two weeks after thesecond immunization, splenocytes are isolated for ELISPOT assays.Immunospot are coated with rat anti-mouse IFN-γ mAb in accordance withmanufacturer instructions, incubated at 4° C. overnight and then blockedwith 200 μl of blocking solution. Subsequently, 2×10⁵ lymphocytes areadded to the wells in triplicate, stimulated with 10 μg/ml ofcorresponding gH or gL peptides or a gH/gL polypeptide mixture (forco-immunization group). After 18 hours, the lymphocytes are discardedand biotin-labeled anti-mouse IFN-γ Ab antibody is added to each welland incubated at 37° C. for 1 h. Next, diluted Streptavidin-HRPconjugate solution is added and incubated at room temperature for 2hours. Finally, the plates are treated with 100 μl of AEC substratesolution and incubated at room temperature for 20 min in the dark. Thereaction is stopped by washing with dematerialized water. Spots arequantified by an ELISPOT reader.

TABLE 2 Human Cytomegalovirus Sequences SEQ Nucleotide ID Sequence NO:Protein Name Protein Sequence (SEQ ID NO:) 32 gi|52139248|ref|MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSE  1 YP_081523.1|PLDKAFHLLLNTYGRPIRFLRENTTQCTYNSS envelopeLRNSTVVRENAISFNFFQSYNQYYVFHMPRC glycoprotein HLFAGPLAEQFLNQVDLTETLERYQQRLNTYA [Human LVSKDLASYRSFSQQLKAQDSLGEQPTTVPPherpesvirus 5] PIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLID ELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKK DQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAF AYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAK RALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQEL YLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRD HSLERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYIVTN QYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAFCQSALLEY DDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSR LLMMSVYALSAIIGIYLLYRMLKTC 33gi|822887470|gb| MPATDTNSTHTTPLHPENQHTLPLHHSTTQP  3 AKI08892.1|HVQTSDKHADKQHRTQMELDAADYAACAQ RL1 protein ARQHLYGQTQPQPHAYPNANPQESAHFCTE[Human NQHQLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW34 gi|822888315|gb| MPATDTNSTHTTPLHPEDQHTLPLQHNTTQP  6 AKI09732.1|HVQTSDKPADKQHRTQMELDAADYAACAQ RL1 protein ARQHLYGQTQ [HumanPQLHAYPNANPQESAHFCTDNQHRLTNLLH herpesvirus 5]NIGEGAALGYPVPRAEIRRGGGDWADSASD FDADCWCMWG RFGTMGRQPVVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHG RRVQLERPSAGEAQARGLLPRIRITPVSTSPRPKAPQPTTSTASHPHATARPDHTLFPVPSTPS ATVHNPRNYAVQLHAETTRTWRWARRGERGAWMPAETFTCPKDKRPW 35 gi|136968|sp|P16 MGRKEMMVRDVPKMVFLISISFLLVSFINCK750.1|GO_HCM VMSKALYNRPWRGLVLSKIGKYKLDQLKLE VA RecName:ILRQLETTISTKYNVSKQPVKNLTMNMTEFP Full = GlycoproteinQYYILAGPIQNYSITYLWFDFYSTQLRKPAK O; Short = gO;YVYSQYNHTAKTITFRPPPCGTVPSMTCLSE Flags: PrecursorMLNVSKRNDTGEQGCGNFTTFNPMFFNVPR WNTKLYVGPTKVNVDSQTIYFLGLTALLLRYAQRNCTHSFYLVNAMSRNLFRVPKYINGT KLKNTMRKLKRKQAPVKEQFEKKAKKTQSTTTPYFSYTTSAALNVTTNVTYSITTAARRVST STIAYRPDSSFMKSIMATQLRDLATWVYTTLRYRQNPFCEPSRNRTAVSEFMKNTHVLIRNE TPYTIYGTLDMSSLYYNETMFVENKTASDSNKTTPTSPSMGFQRTFIDPLWDYLDSLLFLDEI RNFSLRSPTYVNLTPPEHRRAVNLSTLNSLW WWLQ 36gi|583844649|gb| MECNTLVLGLLVLSVVASSNNTSTASTPRPS AHI58989.1|SSTHASTTVKATTVATTSTTTATSTSSTTSAK envelope PGFTTHDPNVMRPHAHNDFYNAHCTSHMYEglycoprotein N LSLSSFAAWWTMLNALILMGAFCIVLRHCCF [Human QNFTATTTKGYherpesvirus 5] 37 gi|136994|sp|P16 MAPSHVDKVNTRTWSASIVFMVLTFVNVSV733.1|GM_HCM HLVLSNFPHLGYPCVYYHVVDFERLNMSAY VA RecName:NVMHLHTPMLFLDSVQLVCYAVFMQLVFL Full = EnvelopeAVTIYYLVCWIKISMRKDKGMSLNQSTRDIS glycoprotein M;YMGDSLTAFLFILSMDTFQLFTLTMSFRLPS Short = gMMIAFMAAVHFFCLTIFNVSMVTQYRSYKRSL FFFSRLHPKLKGTVQFRTLIVNLVEVALGFNTTVVAMALCYGFGNNFFVRTGHMVLAVFVV YAIISIIYFLLIEAVFFQYVKVQFGYHLGAFFGLCGLIYPIVQYDTFLSNEYRTGISWSFGMLFFI WAMFTTCRAVRYFRGRGSGSVKYQALATASGEEVAVLSHHDSLESRRLREEEDDDDDEDF EDA 38 gi|77455773|gb|MSPKDLTPFLTALWLLLGHSRVLRVRAEECC 13 ABA86616.1|EFINVNHPPERCYDFKMCNRFTVALRCPDGE UL128 [HumanVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKL herpesvirus 5]TSCNYNPLYLEADGRIRCGKVNDKAQYLLG AAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ 39 gi|77455773|gb| MSPKDLTPFLTALWLLLGHSRVLRVRAEECC 14ABA86616.1| EFINVNHPPERCYDFKMCNRFTVALRCPDGE UL128 [HumanVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKL herpesvirus 5]TSCNYNPLYLEADGRIRCGKVNDKAQYLLG AAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ 40 gi|822891002|gb| MPATDTNSTHTTPLHPEHHHSTTQPHAQTSD 15AKI12403.1| KHADKQHRTQMELDAADYAACAQARQHL RL1 proteinYGQTQPQLHAYPNANPQESAHFCTENQHQL [Human TNLLHNIGEGAALGYPVPRAEIRRGGGDWAherpesvirus 5] DSASDFDADCWCMWGRFGTMGRQPVVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDG VSVWRQHLVFLLGGHGRRVQLERPSAGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHA TARPDHTLFPVPSTPSATVHNPRNYAVQLHAETTRTWRWARRGERGAWMPAETFTCPKDK RPW 41 gi|52139182|ref|MPATDTNSTHTTPLHPEDQHTLPLHHSTTQP 16 YP_081455.1|HVQTSDKHADKQHRTQMELDAADYAACAQ protein RL1 ARQHLYGQTQPQLHAYPNANPQESAHFRTE[Human NQHQLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW42 gi|52139291|ref| MRLCRVWLSVCLCAVVLGQCQRETAEKND 17 YP_081566.1|YYRVPHYWDACSRALPDQTRYKYVEQLVD envelope proteinLTLNYHYDASHGLDNFDVLKRINVTEVSLLI UL131A SDFRRQNRRGGTNKRTTFNAAGSLAPHARS[Human LEFSVRLFAN herpesvirus 5] 43 gi|52139291|ref|MRLCRVWLSVCLCAVVLGQCQRETAEKND 18 YP_081566.1|YYRVPHYWDACSRALPDQTRYKYVEQLVD envelope proteinLTLNYHYDASHGLDNFDVLKRINVTEVSLLI UL131A SDFRRQNRRGGTNKRTTFNAAGSLAPHARS[Human LEFSVRLFAN herpesvirus 5] 44 gi|52139182|ref|MPATDTNSTHTTPLHPEDQHTLPLHHSTTQP  2 YP_081455.1|HVQTSDKHADKQHRTQMELDAADYAACAQ protein RL1 ARQHLYGQTQPQLHAYPNANPQESAHFRTE[Human NQHQLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW45 gi|52139182|ref| MPATDTNSTHTTPLHPEDQHTLPLHHSTTQP  4 YP_081455.1|HVQTSDKHADKQHRTQMELDAADYAACAQ protein RL1 ARQHLYGQTQPQLHAYPNANPQESAHFRTE[Human NQHQLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW46 gi|822888315|gb| MPATDTNSTHTTPLHPEDQHTLPLQHNTTQP  6 AKI09732.1|HVQTSDKPADKQHRTQMELDAADYAACAQ RL1 protein ARQHLYGQTQPQLHAYPNANPQESAHFCTD[Human NQHRLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPVSTSPRPKAPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW47 gi|52139182|ref| MPATDTNSTHTTPLHPEDQHTLPLHHSTTQP  7 YP_081455.1|HVQTSDKHADKQHRTQMELDAADYAACAQ protein RL1 ARQHLYGQTQPQLHAYPNANPQESAHFRTE[Human NQHQLTNLLHNIGEGAALGYPVPRAEIRRGG herpesvirus 5]GDWADSASDFDADCWCMWGRFGTMGRQP VVTLLLARQRDGLADWNVVRCRGTGFRAHDSEDGVSVWRQHLVFLLGGHGRRVQLERPS AGEAQARGLLPRIRITPISTSPRPKPPQPTTSTASHPHATARPDHTLFPVPSTPSATVHNPRNY AVQLHAETTRTWRWARRGERGAWMPAETF TCPKDKRPW48 hCMV- SSFWTLVQKLIRLTIGK-ERKEE-EEI-  8 gHtruncFLAG,EPPCGQASPPTSSSSPSVSSATYFRHDMAQKP glycoprotein HYPNRWTKRFTYCSTPTGDPSASCVKIPPSVPT Ectodomain TAASVTARSSGKTPSVSTFSKAIINTMYSICLDVFLRVLWRSSF-TR-I- PKPWKDTNRDLTLTRWYPKTWPATDLFRSS- RHKTA-VNSPPLCHRPLTCQYLTFGCHRKPLHTAGQNH IPPQDYTDHTLTRPVSSLMDTIYYSAPSHLVCTKAFTSSTNYVTLK-H-PRTSS-LRCP TTTHPCCLSSAIFHAYFSKRPINATTLYYDKLKNTSSWC-LRKIN-VTLISKTRTFLTPHLTSTT- TSAHYYVTAFTVTPWMYSRAVDVRCWTAAR-KWPSPTH-HCSQQPDKKRPAPKSPSHGP- TARPHSYKYKNL-SPASHKHHHAPRCCCIPRPWTWPNEPFGHRIRSPTSPASYA WSTYSLNRISNISSPNGHYDRSPTLP-NYTKRTWPLFFQPSHAKNSTSWAASSTPCWYI RRRDAKSSS-KRASVHWPSYHTLRSC-LIHTTNTSATCTHPVPVAGDAITRSNASRVSSP MPPSPLPFPPPSPSYLPCNQARWKPSPTCFACRSANPSPR-PSPNTSVIS-QTST- SKVSPTLSPPPS-ARASSSPRRTVKLNAN-RATCIPHTASQWRSTFR-KTAPFAKAPC- NTTTRKASSTSCTCTTRTTSFSPWIPTTKWWSHLRELTTSCF-KTVRY-K- LTSSWTPPTITRTMTISDDNRLEPRWPCFLPLGPPPSPSSPSCTRTPVVFE-SLSGR 49 hCMVgHtrunc6 SSFWTLVQKLIRLTIGK-ERKEE-EEI- 9 XHis, EPPCGQASPPTSSSSPSVSSATYFRHDMAQKP glycoprotein HYPNRWTKRFTYCSTPTGDPSASCVKIPPSVPT Ectodomain-TAASVTARSSGKTPSVSTFSKAIINTMY 6XHis tag SICLDVFLRVLWRSSF-TR-I-PKPWKDTNRDLTLTRWYPKTWPATDLFRSS- RHKTA-VNSPPLCHRPLTCQYLTFGCHRKPLHTAGQNHIPPQDYTDHTLTRPVSSL MDTIYYSAPSHLVCTKAFTSSTNYVTLK-H-PRTSS-LRCP-TTTHPCCLSSAIFHAYFSKR PINATTLYYDKLKNTSSWC-LRKIN-TVTLISKTRTFLTPHLTSTT- TSAHYYVTAFTVTPWMYSRAVD VRCWTAAR-KWPSPTH-HCSQQPDKKRPAPKSPSHGP- TARPHSYKYKNL-SPASHKHHHAPRCCCIPRPWTWPNEPFGHRIRSPTSPASYA WSTYSLNRISNISSPNGHYDRSPTLP-NYTKRTWPLFFQPSHAKNSTSWAASSTPCWYI RRRDAKSSS-KRASVHWPSYHTLRSC-LIHTTNTSATCTHPVPVAGDAITRSNASRVSSP MPPSPLPFPPPSPSYLPCNQARWKPSPTCFACRSANPSPR-PSPNTSVIS-QTST- SKVSPTLSPPPS-ARASSSPRRTVKLNAN-RATCIPHTASQWRSTFR-KTAPFAKAPC- NTTTRKASSTSCTCTTRTTSFSPWIPTTKWWSHLRELTTSCF-KTVRY-K-LTSSWTPPTTITTIT DDNRLEPRWPCFLPLGPPPSPSSPSCTRTPVVFE-SLSGR 50 hCMV_TrgB, SSFWTLVQKLIRLTIGK-ERKEE-EEI- 10glycoprotein B EPPWNPGSGAW-ALTCVSSVWVLRFPHLLLV (ectodomain)ELLLLTVTIPLIRRLLLTLDPVQSLNA- LLPKRSAMVLTRPSTTLPSSTEMWWGSIPPSTPIACVLWPRVRILFALNVISSAPR- SPSMKTWTRASWWSTNATSSRTPLRYESTRRF-RFVVATLTSTPLICWAATRNTW RLLCGRFIISTATVSATVPTAAL-QARFSWLIIGTAMKTKPCN-CPTIIPTPTVPVT- RSRINGTAAAAPGSIVRPVI-IVW-PSLLRAPNILIIFSPLPRVTWLTFLLSTTEPIA MPATLEKTPTSFSFFRTTLSSPTLEDRILR-RPTGWWLFLNVRTR-SPGIYRTKR MSLVNSLSGKPRNAPFVPKPRTRITFLLPK-PPLSYLRSKR-TCPTLRWTAYVMRL- ISYSRFSILHTIKHMKNMETCPSLKPLVVW-CSGKVSSKNLWWNSNVWPTAPV- ILLIIEPKEVQMATMQLIYPTWNRCTIWSTPSCSSPMTRCAVTSTGRWRKSQKPGVWI NGAP-RSSRNSARSTRQPFSRP FTTNRLPRVSWVMSWAWPAA-PSTKPASRCCVI-T- RSRQDAATHDPWSSLISPTARTCSTVNWARTTKSCWATTALRNVSFPASRSSSPGTRPTSTW TTSSNA-LTSAVSPPSTA-SPWISTRWKIPTSGYWNFTRRKSCVPATFLTSKRSCANS TRTSSDNRLEPRWPCFLPLGPPPSPSSPSCTRTPVVFE-SLSGR 51 hCMV_TrgBFL SSFWTLVQKLIRLTIGK-ERKEE-EEI- 11 AG, hCMVEPPWNPGSGAW-SALTCVSSVWVLR glycoproteinBFPHLLLVELLLLTVTIPLIRRLLLTLDPVQSLN ectodomain-A-LLPKRSAMVLTRPSTTLPSSTEMWWG FLAG SIPPSTPIACVLWPRVRILFALNVISSAPR-SPSMKTWTRASWWSTNATSSRTPLRYESTR RF-RFVVATLTSTPLICWAATRNTWRLLCGRFIISTATVSATVPTAAL-QARFSWLII GTAMKTKPCN-CPTIIPTPTVPVT-RSRINGTAAAAPGSIVRPVI-IVW-PSLLRAPN ILIIFSPLPRVTWLTFLLSTTEPIAMPATLEKTPTSFSFFRTTLSSPTLEDRILR-RPTGWWLF LNVRTR-SPGIYRTKRMSLVNSLSGKPRNAPFVPKPRTRITFLLPK- PPLSYLRSKR-TCPTLRWTAYVMRL-ISYSRFSILHTIKHMKNMETCPSLKPLVVW- CSGKVSSKNLWWNSNVWPTAPV-ILLIIEPKEVQMATMQLIYPTWNRCTI WSTPSCSSPMTRCAVTSTGRWRKSQKPGVWINGAP-RSSRNSARSTRQPFSRPFTTNRL PRVSWVMSWAWPAA-PSTKPASRCCVI-T-RSRQDAATHDPWSSLISPTARTCSTVNWARTTK SCWATTALRNVSFPASRSSSPGTRPTSTWTTSSNA-LTSAVSPPSTA-SPWISTRW KIPTSGYWNFTRRKSCVPATFLTSKRSCANSTRTSRITRTMTISDNRLEPRWPCFLPLGPPPSP SSPSCTRTPVVFE-SLSGR 52 hCMV-SSFWTLVQKLIRLTIGK-ERKEE-EEI- 12 TrgB6XHis, EPPWNPGSGAW-SALTCVSSVWVLRhCMV FPHLLLVELLLLTVTIPLIRRLLLTLDPVQSLN glycoproteinA-LLPKRSAMVLTRPSTTLPSSTEMWWGSIP ectodomain- PSTPIACVLWPRVRILFALNVISSAPR-6XHis tag SPSMKTWTRASWWSTNATSSRTPLRYESTR RF-RFVVATLTSTPLICWAATRNTWRLLCGRFIISTATVSATVPTAAL-QARFSWLIIG TAMKTKPCN-CPTIIPTPTVPVT-RSRINGTAAAAPGSIVRPVI-IVW-PSLLRA PNILIIFSPLPRVTWLTFLLSTTEPIAMPATLEKTPTSFSFFRTTLSSPTLEDRILR-RPTGWWLF LNVRTR-SPGIYRTKRMSLVNSLSGKPRNAPFVPKPRTRITFLLPK-PPLSYLRSKR- TCPTLRWTAYVMRL-ISYSRFSILHTIKHMKNMETCPSLKPLVVW- CSGKVSSKNLWWNSNVWPTAPV-ILLIIEPKEVQMATMQLIYPTWNRCTIWST PSCSSPMTRCAVTSTGRWRKSQKPGVWINGAP-RSSRNSARSTRQPFSRPFTTNRLPRVS WVMSWAWPAA-PSTKPASRCCVI-T-RSRQDAATHDPWSSLISPTARTCSTVNWARTTK SCWATTALRNVSFPASRSSSPGTRPTSTWTTSSNA-LTSAVSPPSTA-SPWISTRWKIP TSGYWNFTRRKSCVPATFLTSKRSCANSTRTSSTITTITDNRLEPRWPCFLPLGPPPSPSSPSC TRTPVVFE-SLSGR 55 hCMVMCRRPDCGFSFSPGPVILLWCCLLLPIVSSAA  3 glycoprotein LVSVAPTAAEKVPAECPELTRRCLLGEVFEGD KYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTL LSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPS LFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKY YAGLPPELKQTRVNLPAHSRYGPQAVDAR 56 hCMVMESRIWCLVVCVNLCIVCLGAAVSSSSTRGT  5 glycoprotein BSATHSHHSSHTTSAAHSRSGSVSQRVTSSQT VSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDE GIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQ CYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRE TCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVS DFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSA KMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVF WQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTL RGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTI NQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSL KIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDL EEIMREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVAS VVEGVATFLKNPFGAFTIILVAIAVVIITYLIYTRQRRLCTQPLQNLFPYLVSADGTTVTSGST KDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRAQQ NGTDSLDGRTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENV 57 hCMV UL130 MLRLLLRHHFHCLLLCAVWATPCLASPWST 15LTANQNPSPPWSKLTYSKPHDAATFYCPFLY PSPPRSPLQFSGFQRVSTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQR MPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLESWAHVF RDYSVSFQVRLTFTEANNQTYTFCTHPNLIV 53Ig heavy chain MDWTWILFLVAAATRVHS epsilon-1 signal peptide (IgE HC SP)54 IgGk chain V-III METPAQLLFLLLLWLPDTTG region HAH signal peptide(IgGk SP)

Represents a Stop Sequence

TABLE 3 CMV (Human herpesvirus 5) Amino Acid Sequences GenBank ProteinName Accession Glycoprotein H envelope glycoprotein H [Human herpesvirus5] ACZ79986.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus5] AAA45918.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus5] ACS93310.2 Glycoprotein H envelope glycoprotein H [Human herpesvirus5] AAA45911.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus5] AAA98521.1 Glycoprotein H glycoprotein H [Human herpesvirus 5]BAF44184.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AAA45912.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AKI21335.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AIC80661.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AKI11476.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]ACZ80151.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AHV84023.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AAA45917.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AAA45915.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AFR55394.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AKI14309.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AKI11640.1 Glycoprotein H glycoprotein H [Human herpesvirus 5]BAF44187.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AKI18318.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AHB20043.1 Glycoprotein H envelope glycoprotein H [Human herpesvirus 5]AAA45909.1 Glycoprotein H glycoprotein H [Human herpesvirus 5]BAF44190.1 Glycoprotein H RecName: Full = Envelope glycoprotein H; Short= gH; Flags: Precursor P12824.1 Glycoprotein H envelope glycoprotein H[Human herpesvirus 5] AKI07789.1 Glycoprotein H glycoprotein H [Humanherpesvirus 5] BAF44183.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AGL96664.1 Glycoprotein H glycoprotein H [Humanherpesvirus 5] BAF44189.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AKI08793.1 Glycoprotein H glycoprotein H [Humanherpesvirus 5] BAF44185.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] ADV04392.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AFR56062.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] ACS92000.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AKI15316.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AFR54893.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AHJ86162.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] ACS92165.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] ACT81746.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AKI12305.1 Glycoprotein H envelope glycoprotein H [Humanherpesvirus 5] AKI09634.1 Glycoprotein H glycoprotein H [Humanherpesvirus 5] BAF44191.1 Glycoprotein H RecName: Full = Envelopeglycoprotein H; Short = gH; AltName: P17176.1 Full = Glycoprotein P86;Flags: Precursor [Human herpesvirus 5 strain Towne] Glycoprotein Henvelope glycoprotein H [Human herpesvirus 5] AKI13641.1 Glycoprotein Henvelope glycoprotein H [Human herpesvirus 5] AKI20832.1 Glycoprotein Henvelope glycoprotein H [Human herpesvirus 5] AKI09465.1 Glycoprotein Henvelope glycoprotein H [Human herpesvirus 5] ACS93407.1 Glycoprotein Henvelope glycoprotein H [Human herpesvirus 5] AKI07621.1 Glycoprotein Hglycoprotein H [Human herpesvirus 5] BAF44186.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI22834.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI14981.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI10139.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] ACZ79822.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AAA45910.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AAA45913.1 Glycoprotein Hglycoprotein H [Human herpesvirus 5] BAF44188.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI18822.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AFR56229.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] YP_081523.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI19826.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AAA45914.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI23334.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI14141.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AHB19545.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] ACU83725.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI17318.1 Glycoprotein H envelopeglycoprotein H [Human herpesvirus 5] AKI13975.1 Glycoprotein L RecName:Full = Envelope glycoprotein L; Flags: Precursor [Human Q68672.1herpesvirus 5 (strain 5040)] Glycoprotein L RecName: Full = Envelopeglycoprotein L; Flags: Precursor [Human Q68669.1 herpesvirus 5 (strain2387)] Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI08825.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]ACS92032.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AHJ86194.1 Glycoprotein L RecName: Full = Envelope glycoprotein L;Flags: Precursor P16832.2 Glycoprotein L RecName: Full = Envelopeglycoprotein L; Flags: Precursor [Human Q68671.1 herpesvirus 5 (strain5035)] Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AHB20074.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI12337.1 Glycoprotein L RecName: Full = Envelope glycoprotein L;Flags: Precursor [Human Q68668.1 herpesvirus 5 (strain 1042)]Glycoprotein L envelope glycoprotein L [Human herpesvirus 5] AKI23365.1Glycoprotein L envelope glycoprotein L [Human herpesvirus 5] AKI21032.1Glycoprotein L envelope glycoprotein L [Human herpesvirus 5] YP_081555.1Glycoprotein L RecName: Full = Envelope glycoprotein L; Flags: Precursor[Human Q68670.1 herpesvirus 5 (strain 4654)] Glycoprotein L envelopeglycoprotein L [Human herpesvirus 5] AKI17850.1 Glycoprotein L envelopeglycoprotein L [Human herpesvirus 5] ACZ80183.1 Glycoprotein L envelopeglycoprotein L [Human herpesvirus 5] AKI11508.1 Glycoprotein L envelopeglycoprotein L [Human herpesvirus 5] AKI10171.1 Glycoprotein L RecName:Full = Envelope glycoprotein L; Flags: Precursor [Human Q68673.1herpesvirus 5 (strain 5160)] Glycoprotein L RecName: Full = Envelopeglycoprotein L; Flags: Precursor [Human Q68666.1 herpesvirus 5 strainPT] Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI18350.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AIC80693.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI12003.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI15849.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI13336.1 Glycoprotein L envelope glycoprotein L [Human herpesvirus 5]AKI10840.1 Glycoprotein L RecName: Full = Envelope glycoprotein L;Flags: Precursor Q68667.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] AHV84055.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] AKI07653.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] AFR55425.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] AKI15013.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] ACT81943.1 Glycoprotein L envelope glycoprotein L [Humanherpesvirus 5] AKI21367.1 Glycoprotein L envelope glycoprotein L [Panineherpesvirus 2] NP_612739.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACZ79954.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR56030.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACU83693.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI12106.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI19625.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR55362.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI14613.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI07924.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AIC80127.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHB19512.1 pp150 tegument protein pp150 [Humanherpesvirus 5] YP_081491.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI16116.1 pp150 extended tegument protein pp150 [Humanherpesvirus 5] AII79810.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AIC80629.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR55862.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI10942.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACS91968.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI15451.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACS92133.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR56364.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR54694.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI23468.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI17619.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR55527.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR55193.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR54534.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI18789.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI07588.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI22466.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI20463.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI14780.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI15116.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI14445.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI22633.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI09096.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI13271.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI08760.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR56197.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AFR54861.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACZ80119.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI19960.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI21134.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI11938.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI20128.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI08928.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACZ80284.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI21302.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI12272.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI20967.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI19793.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI23136.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI10106.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI11772.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI08591.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI11443.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI14948.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ADE88040.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI22969.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHJ86130.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACT81879.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI15950.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI15617.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHB19679.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHB19344.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACT81714.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI07756.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI11607.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AIC80295.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI11275.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHB20010.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AIC80463.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI17952.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ACM48022.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI16285.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI09601.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI22299.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AHV83990.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI23301.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AGL96632.1 pp150 tegument protein pp150 [Humanherpesvirus 5] AKI18285.1 pp150 tegument protein pp150 [Humanherpesvirus 5] ADV04360.1 pp65 tegument protein pp65 [Human herpesvirus5] ACZ79994.1 pp65 tegument protein pp65 [Human herpesvirus 5]ACS92173.1 pp65 tegument protein pp65 [Human herpesvirus 5] AKI09642.1pp65 tegument protein pp65 [Human herpesvirus 5] AKI16326.1 pp65tegument protein pp65 [Human herpesvirus 5] ADD39129.1 pp65 tegumentprotein pp65 [Human herpesvirus 5] ACM48061.1 pp65 tegument protein pp65[Human herpesvirus 5] AKI20001.1 pp65 tegument protein pp65 [Humanherpesvirus 5] AKI14149.1 pp65 tegument protein pp65 [Human herpesvirus5] AHB19720.1 pp65 tegument protein pp65 [Human herpesvirus 5]ADV04400.1 pp65 tegument protein pp65 [Human herpesvirus 5] AKI21507.1pp65 tegument protein pp65 [Human herpesvirus 5] AKI15825.1 pp65tegument protein pp65 [Human herpesvirus 5] AKI08299.1 pp65 tegumentprotein pp65 [Human herpesvirus 5] AKI07965.1 pp65 tegument protein pp65[Human herpesvirus 5] AKI22339.1 pp65 tegument protein pp65 [Humanherpesvirus 5] AKI12978.1 pp65 tegument protein pp65 [Human herpesvirus5] AKI11979.1 pp65 tegument protein pp65 [Human herpesvirus 5]AHB19886.1 pp65 tegument protein pp65 [Human herpesvirus 5] YP_081531.1pp65 tegument protein pp65 [Human herpesvirus 5] AKI23010.1 pp65tegument protein pp65 [Human herpesvirus 5] AKI10983.1 pp65 tegumentprotein pp65 [Human herpesvirus 5] AKI10314.1 pp65 tegument protein pp65[Human herpesvirus 5] AFR56070.1 pp65 65K lower matrix phosphoprotein -human cytomegalovirus (strain WMBETW Towne) pp65 mutant UL83 [Humanherpesvirus 5] AAP59842.1 pp65 tegument protein pp65 [Human herpesvirus5] AKI14317.1 pp65 tegument protein pp65 [Human herpesvirus 5]AFR54574.1 pp65 tegument protein pp65 [Human herpesvirus 5] AFR56237.1pp65 tegument protein pp65 [Human herpesvirus 5] AKI18326.1 pp65tegument protein pp65 [Human herpesvirus 5] AKI22842.1 pp65 tegumentprotein PP65 [Human herpesvirus 5] AHV84031.1 pp65 tegument protein[synthetic construct] AAT68258.1 pp65 phosphorylated matrix protein(pp65) [Human herpesvirus 5] AAA45996.1 pp65 tegument protein pp65[Panine herpesvirus 2] NP_612716.1 pp65 tegument protein pp65 [Humanherpesvirus 5] ADJ68256.1 pp65 tegument protein pp65 [Human herpesvirus5] ADJ68266.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]ACT81935.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]YP_081547.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]ACM48077.1 UL100 (gM) RecName: Full = Envelope glycoprotein M; Short =gM P16733.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]AKI18175.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]AFR54590.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]AKI20017.1 UL100 (gM) envelope glycoprotein M [Human herpesvirus 5]AKI09994.1 UL100 (gM) UL100 [Human herpesvirus 5] AAS48986.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI20856.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI14333.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AHB19736.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AGT36389.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] ACZ80175.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI18009.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI23358.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AHV84047.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] ACS92024.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI10999.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AKI16173.1 UL100 (gM)envelope glycoprotein M [Human herpesvirus 5] AFR54917.1 UL100 (gM)UL100 [Human herpesvirus 5] ABV71622.1 UL100 (gM) envelope glycoproteinM [Human herpesvirus 5] AKI13999.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI12329.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI21523.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI18342.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI09658.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI07813.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI18846.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AFR55081.1 UL100 (gM) envelope glycoprotein M[Human herpesvirus 5] AKI17342.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ACT81950.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI07996.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31361.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI09840.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI12010.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31390.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR55598.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI08832.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27071.1 UL123 regulatory protein IE1[Human herpesvirus 5] AHB19584.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI18861.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31419.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI23372.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI16357.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI10512.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI19028.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI11347.1 UL123 72 kDa immediate-early 1 protein [Humanherpesvirus 5] ACT34667.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB84698.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27072.1 UL123 regulatory protein IE1[Human herpesvirus 5] AKI22873.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI20200.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI12677.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AHV84062.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR55096.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR54932.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI22205.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR55264.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI18357.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI17188.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI12841.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI09673.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI21537.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI20871.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AGL96703.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31477.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI21374.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ACZ80025.1 UL123 72 kDa immediate-early 1 protein [Humanherpesvirus 5] ACT34666.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI20032.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31303.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AIC80700.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB84746.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27084.1 UL123 regulatory protein IE1[Human herpesvirus 5] ADV04431.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI11515.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR56435.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27074.1 UL123 regulatory protein IE1[Human herpesvirus 5] AKI14180.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI07828.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AHB19751.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB84818.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI18526.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI14014.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ACT81785.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB44102.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI15187.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27092.1 UL123 immediate earlytranscriptional regulator [Human herpesvirus 5] ACL27056.1 UL123regulatory protein IE1 [Human herpesvirus 5] AKI18024.1 UL123 regulatoryprotein IE1 [Human herpesvirus 5] AKI14517.1 UL123 regulatory proteinIE1 [Human herpesvirus 5] ADE88106.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI15355.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI10178.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB84722.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ADB84650.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AFR56268.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] YP_081562.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI22538.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AHB19917.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31332.1 UL123 RecName: Full = 55 kDa immediate-earlyprotein 1; Short = IE1 P13202.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AAR31448.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI14852.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] AKI14348.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27066.1 UL123 immediate earlytranscriptional regulator [Human herpesvirus 5] ACL27058.1 UL123immediate early transcriptional regulator [Human herpesvirus 5]ACL27086.1 UL123 regulatory protein IE1 [Human herpesvirus 5] AKI16022.1UL123 regulatory protein IE1 [Human herpesvirus 5] AIC80534.1 UL123immediate early transcriptional regulator [Human herpesvirus 5]ACL27094.1 UL123 immediate early transcriptional regulator [Humanherpesvirus 5] ACL27093.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27073.1 UL123 pp65/IE1 fusion protein[synthetic construct] ABQ23593.1 UL123 regulatory protein IE1 [Humanherpesvirus 5] ACS92204.1 UL123 RecName: Full = 55 kDa immediate-earlyprotein 1; Short = IE1 [Human P03169.1 herpesvirus 5 strain Towne] UL123regulatory protein IE1 [Human herpesvirus 5] ADB84794.1 UL123 majorimmediate-early protein [Human herpesvirus 5] AAA45979.1 UL123 immediateearly transcriptional regulator [Human herpesvirus 5] ACL27059.1 UL123regulatory protein IE1 [Human herpesvirus 5] AKI21039.1 UL123 immediateearly transcriptional regulator [Human herpesvirus 5] ACL27065.1 UL123immediate early transcriptional regulator [Human herpesvirus 5]ACL27087.1 UL123 regulatory protein IE1 [Human herpesvirus 5] AAR31504.1UL123 immediate early transcriptional regulator [Human herpesvirus 5]ACL27082.1 UL123 immediate early transcriptional regulator [Humanherpesvirus 5] ACL27055.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27081.1 UL123 regulatory protein IE1[Human herpesvirus 5] AKI12344.1 UL123 immediate early transcriptionalregulator [Human herpesvirus 5] ACL27089.1 UL123 immediate earlytranscriptional regulator [Human herpesvirus 5] ACL27062.1 UL123immediate early transcriptional regulator [Human herpesvirus 5]ACL27057.1 UL128 envelope protein UL128 [Human herpesvirus 5] AAR31451.1UL128 UL128 [Human herpesvirus 5] ABA86617.1 UL128 envelope proteinUL128 [Human herpesvirus 5] AAR31335.1 UL128 envelope protein UL128[Human herpesvirus 5] ADV04433.1 UL128 UL128 [Human herpesvirus 5]ADF30829.1 UL128 envelope protein UL128 [Human herpesvirus 5] ACS92206.1UL128 envelope protein UL128 [Human herpesvirus 5] ADB84652.1 UL128envelope protein UL128 [Human herpesvirus 5] AHJ86203.1 UL128 envelopeprotein UL128 [Human herpesvirus 5] AAO11759.2 UL128 envelope proteinUL128 [Human herpesvirus 5] AKI07662.1 UL128 UL128 [Human herpesvirus 5]ABA86608.1 UL128 envelope protein UL128 [Human herpesvirus 5] AKI12512.1UL128 envelope protein UL128 [Human herpesvirus 5] AKI21705.1 UL128envelope protein UL128 [Human herpesvirus 5] AKI20034.1 UL128 RecName:Full = Uncharacterized protein UL128 P16837.2 UL128 UL128 [Humanherpesvirus 5] ABA86623.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI16857.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI18528.1 UL128 envelope protein UL128 [Humanherpesvirus 5] ADB84820.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AAR31422.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AGL96705.1 UL128 envelope protein UL128 [Humanherpesvirus 5] ACT81952.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI18359.1 UL128 UL128 [Human herpesvirus 5] AAO11775.2UL128 UL128 [Human herpesvirus 5] ABA86605.1 UL128 UL128 [Humanherpesvirus 5] ADF30833.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI11182.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI15691.1 UL128 UL128 [Human herpesvirus 5] ABA86622.1UL128 UL128 [Human herpesvirus 5] ADF30832.1 UL128 UL128 [Humanherpesvirus 5] ABA86616.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AAO11755.2 UL128 envelope protein UL128 [Humanherpesvirus 5] AFR55266.1 UL128 UL128 [Human herpesvirus 5] ABA86618.1UL128 envelope protein UL128 [Human herpesvirus 5] ADB84700.1 UL128UL128 [Human herpesvirus 5] ADE62337.1 UL128 UL128 [Human herpesvirus 5]ADF30837.1 UL128 UL128 [Human herpesvirus 5] ABA86604.1 UL128 envelopeprotein UL128 [Human herpesvirus 5] AKI21208.1 UL128 UL128 [Humanherpesvirus 5] ABA86609.1 UL128 envelope protein UL128 [Humanherpesvirus 5] AKI10514.1 UL128 truncated UL128 protein [Humanherpesvirus 5] ADG36331.1 UL128 HCMVUL128 [Human herpesvirus 5]CAA35330.1 UL130 UL130 [Human herpesvirus 5] ABA86653.1 UL130 UL130[Human herpesvirus 5] ABA86666.1 UL130 UL130 [Human herpesvirus 5]ABA86652.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]YP_081565.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI08835.1 UL130 UL130 [Human herpesvirus 5] AAY33781.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] ACS92042.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AKI10515.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AKI07663.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AHB19754.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AFR55435.1 UL130 UL130 [Humanherpesvirus 5] AAY33778.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AKI18864.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AIC80537.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] ADB44105.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] ADB84797.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AKI22373.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AGL96706.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AKI22042.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AHJ86204.1 UL130 RecName: Full = Uncharacterized proteinUL130 P16772.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI20706.1 UL130 UL130 [Human herpesvirus 5] ABA86662.1 UL130 UL130[Human herpesvirus 5] ABA86665.1 UL130 UL130 [Human herpesvirus 5]ABA86659.1 UL130 UL130 [Human herpesvirus 5] ADF30831.1 UL130 orf UL130[Human herpesvirus 5] AAA85889.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AKI11183.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AKI19031.1 UL130 UL130 [Human herpesvirus 5]ADE62342.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AFR55099.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AAR31336.1 UL130 UL130 [Human herpesvirus 5] ABA86654.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AKI17191.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] ADB84701.1 UL130 UL130 [Humanherpesvirus 5] ADF30838.1 UL130 envelope glycoprotein UL130 [Humanherpesvirus 5] AKI15859.1 UL130 UL130 [Human herpesvirus 5] ABA86651.1UL130 envelope glycoprotein UL130 [Human herpesvirus 5] AFR55267.1 UL130envelope glycoprotein UL130 [Human herpesvirus 5] ACS92207.1 UL130envelope glycoprotein UL130 [Human herpesvirus 5] AAR31307.1 UL130envelope glycoprotein UL130 [Human herpesvirus 5] AKI15358.1 UL130envelope glycoprotein UL130 [Human herpesvirus 5] AKI21042.1 UL130envelope glycoprotein UL130 [Human herpesvirus 5] AKI16360.1 UL130 UL130[Human herpesvirus 5] ABA86661.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AHB19920.1 UL130 UL130 [Human herpesvirus 5]ABV71640.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI23375.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI08333.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI21377.1 UL130 envelope glycoprotein UL130 [Human herpesvirus 5]AKI16526.1 UL130 UL130 [Human herpesvirus 5] ADE62336.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AKI14017.1 UL130 envelopeglycoprotein UL130 [Human herpesvirus 5] AKI09843.1 UL130 mutant fusionprotein [Human herpesvirus 5] ADE62322.1 UL130 envelope glycoproteinUL130 [Human herpesvirus 5] AKI12013.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AKI20371.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] ACZ81666.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AAR31365.1 UL130 envelope glycoprotein UL130[Human herpesvirus 5] AAO11754.1 UL131A envelope protein UL131A [Humanherpesvirus 5] YP_081566.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AKI12514.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AKI11683.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AAO11766.1 UL131A UL131A [Human herpesvirus 5] ABA86643.1UL131A UL131A [Human herpesvirus 5] ADE62341.1 UL131A truncated envelopeprotein UL131A [Human herpesvirus 5] ADV04435.1 UL131A envelope proteinUL131A [Human herpesvirus 5] AFR56272.1 UL131A envelope protein UL131A[Human herpesvirus 5] AKI11018.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AHB19755.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AKI12348.1 UL131A UL131a protein [Human herpesvirus 5]ADG36333.1 UL131A UL131A [Human herpesvirus 5] ABA86640.1 UL131Aenvelope protein UL131A [Human herpesvirus 5] AKI08836.1 UL131A UL131A[Human herpesvirus 5] ABA86639.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AKI10182.1 UL131A envelope protein UL131A [Humanherpesvirus 5] ADB84774.1 UL131A envelope protein UL131A [Humanherpesvirus 5] ADB84822.1 UL131A UL131A [Human herpesvirus 5] ADF30839.1UL131A UL131A [Human herpesvirus 5] ABA86648.1 UL131A UL131A [Humanherpesvirus 5] ABA86635.1 UL131A envelope protein UL131A [Humanherpesvirus 5] AFR55436.1 UL131A UL131A [Human herpesvirus 5] ABA86637.1UL131A UL131A [Human herpesvirus 5] ABA86644.1 UL131A UL131A [Humanherpesvirus 5] ABA86647.1 UL131A UL131A [Human herpesvirus 5] ABA86629.1UL131A UL131A [Human herpesvirus 5] ABA86630.1 UL131A UL131A [Humanherpesvirus 5] ABA86646.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] ACS91991.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] AKI12129.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] ACZ79977.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] AFR55216.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] AKI22656.1 UL55 (gB) glycoprotein B [Human herpesvirus 5]AAA45934.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AFR54884.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI22156.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI14299.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]ADV04383.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI20990.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI09624.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]ADD39116.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]ACT81737.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI11131.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI17642.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AIC80652.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AFR55719.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]AKI09288.1 UL55 (gB) glycoprotein B [Human herpesvirus 5] AAA45930.1UL55 (gB) envelope glycoprotein B [Human herpesvirus 5] AKI12960.1 UL55(gB) glycoprotein B [Human herpesvirus 5] AAA45926.1 UL55 (gB)glycoprotein B [Human herpesvirus 5] AAA45925.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AII80437.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AKI22824.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AHV84013.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AKI07947.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AFR54557.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AHB19702.1 UL55 (gB) RecName: Full= Envelope glycoprotein B; Short = gB; Flags: Precursor P06473.1 UL55(gB) glycoprotein B [Human herpesvirus 5] ADB92600.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] ADE88063.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AHJ86153.1 UL55 (gB) envelopeglycoprotein B [Human herpesvirus 5] AFR55885.1 UL55 (gB) UL55 [Humanherpesvirus 5] ABV71586.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] ACS92156.1 UL55 (gB) envelope glycoprotein B [Humanherpesvirus 5] AKI23491.1 UL55 (gB) RecName: Full = Envelopeglycoprotein B; Short = gB; Contains: P13201.1 RecName: Full =Glycoprotein GP55; Flags: Precursor UL55 (gB) glycoprotein B [Humanherpesvirus 5] ABQ23592.1 UL55 (gB) glycoprotein B [Human herpesvirus 5]AAB07485.1 UL55 (gB) envelope glycoprotein B [Human herpesvirus 5]ACM48044.1 UL55 (gB) glycoprotein B [Human herpesvirus 5] AAA45928.1UL55 (gB) envelope glycoprotein B [Human herpesvirus 5] ACS32370.1 UL55(gB) envelope glycoprotein B [Human herpesvirus 5] AKI19983.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI13294.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AFR55048.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI19483.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] YP_081514.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI20319.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AHB20033.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI23324.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI13965.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] ACS93398.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI08783.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AFR55550.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AKI19648.1 UL55 (gB)envelope glycoprotein B [Human herpesvirus 5] AGL96655.1 UL55 (gB)glycoprotein B [Human herpesvirus 5] AAA45932.1 UL55 (gB) glycoprotein B[Human herpesvirus 5] AAA45933.1 UL55 (gB) glycoprotein B [Gorillagorilla cytomegalovirus 2.1] ACT68391.2 UL55 (gB) glycoprotein B [Humanherpesvirus 5] AAA45931.1 UL55 (gB) glycoprotein B [Human herpesvirus 5]AAA45923.1 UL55 (gB) glycoprotein gB precursor [synthetic construct]AAT68257.1 UL55 (gB) glycoprotein B [Human herpesvirus 5] AAA45924.1UL55 (gB) glycoprotein B [Human herpesvirus 5] AAA45935.1 UL73 (gN)structural glycoprotein gpUL73 [Human herpesvirus 5] AAM82374.1 UL73(gN) structural glycoprotein gpUL73 [Human herpesvirus 5] AAG23509.1UL73 (gN) structural glycoprotein gpUL73 [Human herpesvirus 5]AAM82416.1 UL73 (gN) envelope glycoprotein gpUL73 [Human herpesvirus 5]AAO24877.1 UL73 (gN) envelope glycoprotein N [Human herpesvirus 5]ADE20136.1 UL73 (gN) envelope glycoprotein N [Human herpesvirus 5]YP_081521.1 UL73 (gN) glycoprotein N [Human herpesvirus 5] ACI45834.1UL73 (gN) envelope glycoprotein gpUL73 [Human herpesvirus 5] AAO27562.1UL73 (gN) glycoprotein N [Human herpesvirus 5] ACI45816.1 UL73 (gN)envelope glycoprotein N [Human herpesvirus 5] ACS93313.1 UL73 (gN)envelope glycoprotein N [Human herpesvirus 5] AKI07618.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ADC32373.1 UL73 (gN) structuralglycoprotein gpUL73 [Human herpesvirus 5] AAG23521.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ADH42929.1 UL73 (gN) glycoprotein N[Human herpesvirus 5] ADC32376.1 UL73 (gN) glycoprotein N [Humanherpesvirus 5] ADH42919.1 UL73 (gN) glycoprotein N [Human herpesvirus 5]ACI45808.1 UL73 (gN) envelope glycoprotein gpUL73 [Human herpesvirus 5]AAO24851.1 UL73 (gN) glycoprotein N [Human herpesvirus 5] ABY48941.1UL73 (gN) structural glycoprotein gpUL73 [Human herpesvirus 5]AAG23512.1 UL73 (gN) structural glycoprotein gpUL73 [Human herpesvirus5] AAM82399.1 UL73 (gN) UL73 [Human herpesvirus 5] ABZ04151.1 UL73 (gN)envelope glycoprotein gpUL73 [Human herpesvirus 5] AAO24895.1 UL73 (gN)structural glycoprotein gpUL73 [Human herpesvirus 5] AAM82420.1 UL73(gN) structural glycoprotein UL73 [Human herpesvirus 5] AAL77782.1 UL73(gN) glycoprotein N [Human herpesvirus 5] ADH42921.1 UL73 (gN)structural glycoprotein gpUL73 [Human herpesvirus 5] AAM82396.1 UL73(gN) envelope glycoprotein gpUL73 [Human herpesvirus 5] AAO24881.1 UL73(gN) envelope glycoprotein gpUL73 [Human herpesvirus 5] AAO24889.1 UL73(gN) envelope glycoprotein gpUL73 [Human herpesvirus 5] AAO24892.1 UL73(gN) glycoprotein N [Human herpesvirus 5] ABY48942.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ACI45800.1 UL73 (gN) envelopeglycoprotein gpUL73 [Human herpesvirus 5] AAO27565.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ABY48936.1 UL73 (gN) glycoprotein N[Human herpesvirus 5] ABY48935.1 UL73 (gN) structural glycoproteingpUL73 [Human herpesvirus 5] AAM82375.1 UL73 (gN) structuralglycoprotein gpUL73 [Human herpesvirus 5] AAM82403.1 UL73 (gN) envelopeglycoprotein N [Human herpesvirus 5] AHB19542.1 UL73 (gN) envelopeglycoprotein N [Human herpesvirus 5] AKI23166.1 UL73 (gN) structuralglycoprotein gpUL73 [Human herpesvirus 5] AAM82412.1 UL73 (gN) envelopeglycoprotein gpUL73 [Human herpesvirus 5] AAO24836.1 UL73 (gN) UL73[Human herpesvirus 5] ABZ04148.1 UL73 (gN) envelope glycoprotein N[Human herpesvirus 5] ACS93153.1 UL73 (gN) envelope glycoprotein N[Human herpesvirus 5] AGT36363.1 UL73 (gN) structural glycoproteingpUL73 [Human herpesvirus 5] AAG23511.1 UL73 (gN) glycoprotein N [Humanherpesvirus 5] ADH42925.1 UL73 (gN) glycoprotein N [Human herpesvirus 5]ACI45830.1 UL73 (gN) structural glycoprotein gpUL73 [Human herpesvirus5] AAG23510.1 UL73 (gN) glycoprotein N [Human herpesvirus 5] ACI45798.1UL73 (gN) structural glycoprotein UL73 [Human herpesvirus 5] AAL77762.1UL73 (gN) glycoprotein N [Human herpesvirus 5] ADH42926.1 UL73 (gN)structural glycoprotein UL73 [Human herpesvirus 5] AAL77766.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ACI45823.1 UL73 (gN) structuralglycoprotein gpUL73 [Human herpesvirus 5] AAM82378.1 UL73 (gN)structural glycoprotein gpUL73 [Human herpesvirus 5] AAM82379.1 UL73(gN) UL73 [Human herpesvirus 5] ABZ04149.1 UL73 (gN) structuralglycoprotein UL73 [Human herpesvirus 5] AAL77764.1 UL73 (gN)glycoprotein N [Human herpesvirus 5] ACI45835.1 UL73 (gN) glycoprotein N[Human herpesvirus 5] ACI45825.1 UL73 (gN) glycoprotein N [Humanherpesvirus 5] ADH42931.1 UL73 (gN) envelope glycoprotein N [Humanherpesvirus 5] ACS93218.1 UL73 (gN) structural glycoprotein gpUL73[Human herpesvirus 5] AAM82408.1 UL73 (gN) glycoprotein N [Humanherpesvirus 5] ACI45831.1 UL73 (gN) envelope glycoprotein gpUL73 [Humanherpesvirus 5] AAO27561.1 UL73 (gN) glycoprotein N [Human herpesvirus 5]ACI45826.1 UL74 (gO) UL74 protein [Human herpesvirus 5] AAN40064.1 UL74(gO) envelope glycoprotein O [Human herpesvirus 5] AKI16316.1 UL74 (gO)envelope glycoprotein O [Human herpesvirus 5] ACS93259.1 UL74 (gO) UL74protein [Human herpesvirus 5] AAN40079.1 UL74 (gO) envelope glycoproteinO [Human herpesvirus 5] AKI18316.1 UL74 (gO) glycoprotein O [Humanherpesvirus 5] ABY48961.1 UL74 (gO) glycoprotein O [Human herpesvirus 5]ABY48960.1 UL74 (gO) glycoprotein O [Human herpesvirus 5] ABY48959.1UL74 (gO) envelope glycoprotein O [Human herpesvirus 5] ACS93169.1 UL74(gO) UL74 protein [Human herpesvirus 5] AAN40044.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] ACS93340.1 UL74 (gO) RecName: Full= Glycoprotein O; Short = gO; Flags: Precursor P16750.1 UL74 (gO) UL74protein [Human herpesvirus 5] AAN40046.1 UL74 (gO) UL74 protein [Humanherpesvirus 5] AAN40054.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AKI08959.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AKI20327.1 UL74 (gO) UL74 protein [Human herpesvirus 5]AAN40071.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]AHB19710.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]AKI07787.1 UL74 (gO) UL74 protein [Human herpesvirus 5] AAN40043.1 UL74(gO) UL74 protein [Human herpesvirus 5] AAN40078.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] ACS93309.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] ACS93234.1 UL74 (gO) UL74 protein[Human herpesvirus 5] AAN40040.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI19491.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI16979.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI20998.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI23000.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI10806.1 UL74 (gO) UL74 protein [Humanherpesvirus 5] AAN40073.1 UL74 (gO) UL74 protein [Human herpesvirus 5]AAN40057.1 UL74 (gO) UL74 protein [Human herpesvirus 5] AAN40050.1 UL74(gO) glycoprotein O [Human herpesvirus 5] ABY48952.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] AKI09296.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] ACS93149.1 UL74 (gO) envelopeglycoprotein O [Human herpesvirus 5] AKI14979.1 UL74 (gO) UL74 protein[Human herpesvirus 5] AAN40060.1 UL74 (gO) glycoprotein O [Humanherpesvirus 5] ABY48954.1 UL74 (gO) glycoprotein O [Human herpesvirus 5]ABY48955.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]ACS93219.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]ACS93164.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]YP_081522.1 UL74 (gO) glycoprotein O [Human herpesvirus 5] ABY48956.1UL74 (gO) envelope glycoprotein O [Human herpesvirus 5] AKI11474.1 UL74(gO) UL74 protein [Human herpesvirus 5] AAN40039.1 UL74 (gO) UL74protein [Human herpesvirus 5] AAN40041.1 UL74 (gO) envelope glycoproteinO [Human herpesvirus 5] ACS93154.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] ACT81745.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] ACS92164.1 UL74 (gO) UL74 protein [Humanherpesvirus 5] AAN40052.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AHJ86161.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] ACS93204.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AKI15314.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] ACZ80315.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AKI23332.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] ACU83724.1 UL74 (gO) UL74 protein [Human herpesvirus 5]AAN40047.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]AHV84021.1 UL74 (gO) UL74 protein [Human herpesvirus 5] AAN40056.1 UL74(gO) envelope glycoprotein O [Human herpesvirus 5] AKI22164.1 UL74 (gO)envelope glycoprotein O [Human herpesvirus 5] ACS93189.1 UL74 (gO) UL74protein [Human herpesvirus 5] AAN40074.1 UL74 (gO) envelope glycoproteinO [Human herpesvirus 5] AKI18820.1 UL74 (gO) envelope glycoprotein O[Human herpesvirus 5] AKI07619.1 UL74 (gO) UL74 protein [Humanherpesvirus 5] AAN40072.1 UL74 (gO) envelope glycoprotein O [Humanherpesvirus 5] AKI19991.1 UL74 (gO) UL74 protein [Human herpesvirus 5]AAN40062.1 UL74 (gO) envelope glycoprotein O [Human herpesvirus 5]AKI10471.1 UL74 (gO) UL74 protein [Human herpesvirus 5] AAN40042.1 UL74(gO) glycoprotein O [Human herpesvirus 5] AAT91377.1 UL74 (gO)glycoprotein O [Human herpesvirus 5] ACI45857.1 UL74 (gO) UL74 protein[Human herpesvirus 5] AAP88253.1 RecName: Full = Large structuralphosphoprotein; AltName: Full = 150 P08318.1 kDa matrix phosphoprotein;AltName: Full = 150 kDa phosphoprotein; Short = pp150; AltName: Full =Basic phosphoprotein; Short = BPP; AltName: Full = Phosphoprotein UL32;AltName: Full = Tegument protein UL32 UL32 [Human herpesvirus 5]ABV71562.1 UL32 [Human herpesvirus 5] AAG31644.1 UL32 [Human herpesvirus5] AAS48942.1 UL83 [Human herpesvirus 5] ABV71605.1 RecName: Full = 65kDa phosphoprotein; Short = pp65; AltName: P06725.2 Full = 65 kDa matrixphosphoprotein; AltName: Full = Phosphoprotein UL83; AltName: Full =Tegument protein UL83 RecName: Full = 65 kDa phosphoprotein; Short =pp65; AltName: P18139.2 Full = 64 kDa matrix phosphoprotein; Short =pp64; AltName: Full = GP64; AltName: Full = Phosphoprotein UL83;AltName: Full = Tegument protein UL83 [Human herpesvirus 5 strain Towne]HCMVUL115 [Human herpesvirus 5] CAA35317.1 truncated UL115 protein[Human herpesvirus 5] ADG34192.1

Example 22: Expression of mRNA Vaccine Constructs Encoding the hCMVPentameric Complex in HeLa Cells

Expression of mRNA vaccine constructs encoding the subunits of the hCMVpentameric complex, including gH, gL, UL128, UL130, and UL131A wastested (FIG. 1B). mRNAs encoding each subunit were mixed at agH:gL:UL128:UL130:UL131A ratio of 4:2:1:1:1. The total amount of mRNAused for transfecting HeLa cells was 2 μg. The transfected HeLa cellswere incubated for 24 hours before they were analyzed usingfluorescence-activated cell sorting (FACS) on a flow cytometer for thesurface expression of the pentameric complex subunits as well as thecomplete pentamer (FIGS. 2A-2D).

Antibodies specific for gH, UL128, the UL128/130/131A complex, or thecomplete pentamer were used for the detection of surface expression ofthe proteins. Surface expression of gH, UL128, the UL128/130/131Acomplex, and the complete pentameric complex were detected in HeLa cells(FIGS. 2A-2D).

Different combinations of the mRNAs encoding the pentameric subunitswere also tested to determine whether all of the core subunits were needfor the surface expression of the complete pentameric complex (FIGS.3A-3B). The experiments were carried out as described above with theindicated mRNA combinations. An antibody specific for the completepentameric complex was used (8121). The results show that the pentamericcomplex does not express on the cell surface in the absence of any ofUL128, UL130, or UL131A.

Next, the surface expression of the gH glycoprotein with or without gLwas tested. The experiments were carried out as described above usingmRNA constructs encoding gH, gH and gL, or constructs encoding thepentameric complex. An antibody specific for gH (3G16) was used. Theresults showed that expression of gH alone does not lead to gHexpression on the cell surface. However, when gH was complexed with gL,a similar level of gH was detected on the surface of the HeLa cells aswhen all subunits in the pentameric complex were expressed (FIGS.4A-4B).

The intracellular and surface expression of gB was also tested usingantibodies specific for gB. FIG. 5A shows intracellular gB expression.The surface expression of gB was measured by FACS on a flow cytometerand surface expression of gB was detected (FIG. 5B). The quantificationof gB surface expression is shown in FIG. 5C. Further, an immunoblotconducted on cell lysates from HeLa cells transfected with mRNAconstructs encoding gB is shown in FIG. 5D. Untransfected HeLa celllysates were used as a negative control and reconstituted full-length gBprotein was used as a positive control. As shown in FIG. 5D, middlelane, both full-length gB (the precursor) and the mature gB afterproteolytic cleavage were detected.

Example 23: High Titers of Anti-Pentameric Antibodies FollowingImmunization with hCMV Pentameric Complex mRNA Vaccine Constructs

The immunogenicity of candidate hCMV mRNA vaccine constructs encodingthe pentameric complex subunits and/or the gB antigen was tested inmice. The immunization schedule and mRNA fomulations ares shown in Table4 below.

Mice were divided into groups (5 mice per group) and vaccinated on day0, 21, and 42 via intramuscular (IM) routes. One group of mice wasvaccinated with empty lipid nanoparticles (LNP) as a control. Othergroups of mice received hCMV mRNA vaccine constructs encoding thepentameric complex, the gB antigen, both the pentameric complex and gBantigen, or either the pentameric protein complex or the gB proteinantigen combined with MF59. When mRNA vaccine constructions were given,different preparation procedures were used. The “pre-mix” mRNAs werepre-mixed and then formulated, while the “post-mix” mRNAs wereindividually formulated and then mixed. The mRNAs encoding all thesubunits of the pentameric complex were formulated with different ratiosas shown in Table 4: gH-gL-UL128-UL130-UL131A was 4:2:1:1:1 or1:1:1:1:1. gB+pentamer was formulated at 1:1:1:1:1:1. The dose schedulesused are indicated in Table 4.

Mice sera were collected from each mouse on days −1 (pre-dos), 20, 41,62, and 84. Individual bleeds from all time points were tested via ELISAassay carried out on plates coated with hCMV pentamers. Serum samplestypically were diluted 1:100 for the assay. Incubation and washingprotocols were performed using routine methods. Data was read at 450 nmwavelength. Data was reported and plotted (FIGS. 6 and 7). FIG. 6 showsthat anti-pentamer-specific IgG were induced by hCMV mRNA vaccineconstructs. However, little or no boosting was observed after the 3^(rd)immunization. IgG response was maintained from 6-9 weeks following asingle immunization. Adding mRNAs encoding gB to mRNAs encoding thepentameric complex subunits did not interefere with anti-pentameric IgGproduction. Different molar ratios of the mRNAs encoding differentpentameric complex subunits did not lead to different IgG inductionlevels. FIG. 7 shows that the mRNA vaccine constructs encoding gBinduced anti-gB IgG response. IgG titers were similar for gB mRNAcompared to gB protein/MF59 at 10 μg dose after three immunizations. Aboost response was observed after the 3^(rd) immunization of gB mRNAs orantigens. Adding mRNAs encoding the pentameric complex subunits to mRNAsencoding gB did not interefere with anti-gB IgG production.

Example 24: Immunization with hCMV Pentameric Complex mRNA ElicitsHighly Potent Neutralizing Antibodies in Mice

Neutralization assays were conducted in epithelial cell line ARPE-19infected with hCMV clinical isolate VR1814 were conducted. Mice wereimmunized according to the methods in Example 23. Mouse serum sampleswere collected 3 weeks after the second immunization (on day 41). Micesera collected from mice immunized with 3 μg of hCMV mRNA pentamericvaccine constructs were diluted (1:25600) and added to the infectedcells. The cells were stained for hCMV IE1 protein (as an indication ofthe presence of hCMV in the cells). Results showed that serum from miceimmunized with 3 μg of hCMV pentameric mRNA vaccine constructs were ableto neutralize the hCMV in ARPE-19 cells, while the controls of humanseropositive serum or no serum did not neutralize the hCMV in ARPE-19cells (FIG. 8).

The hCMV neutralization titers of mouse serum measured in ARPE-19 cellsinfected with clinical hCMV isolate strain VR1814 are shown in FIG. 9and Table 5.

TABLE 4 Immunization schedule Group Vaccine Route Dose schedule N 1Pentamer IM d 0 (10 ug) 5 (*4:2:1:1:1, pre- mix), Equimolar 2 PentamerIM d 0, d 21, d 42 5 (4:2:1:1:1, pre- (10 ug, 3 ug, mix) 1 ug) 3Pentamer IM d 0 (10 ug) 5 (4:2:1:1:1), post-mix) 4 Pentamer IM d 0, d21, d 42 5 (4:2:1:1:1, post- (10 ug, 3 ug, 1 ug) mix) 5 Pentamer IM d 0,d 21, d 42 5 (1:1:1:1:1, pre- (10 ug) mex), Equal conc 6 gB IM d 0, d21, d 42 5 (10 ug) 7 gB + Pentamer IM d 0 (12 ug) 5 (1:1:1:1:1:1,pre-mix) 8 gB + Pentamer IM d 0, d 21, d 42 5 (1:1:1:1:1:1, (12 ug)pre-mix) 9 Pentamer IM d 21, d 42 (10 ug) 5 protein/MF59 10 gB IM d 0, d21, d 42 5 protein/MF59 (10 ug) 11 Empty LNP IM d 0, d 21, d 42 5

TABLE 5 hCMV neutralization titers of mouse serum measured in ARPE-19cells infected with clinical isolate VR1814 # of Dose NT50 Formulationdoses (ug) Titer Pentamer, 2 10 >2E4 Pre-mix (Equimoiar) Pentamer, 23 >2E4 Pre-mix (Equimolar) Pentamer, 2 10 >2E4 Post-mix (Equimolar)Pentamer, 7 3 >2E4 Post-mix (Equimolar) Pentamer + gB 2 12 >2E4 (Equalconc)

Example 25: Second Generation hCMV Pentameric Complex mRNA VaccineConstructs

HCMV pentameric complex mRNA vaccine constructs were modified to producesecond generation mRNA constructs. The nucleotide sequences of thesecond generation mRNA constructs and the encoded amino acid sequencesare provided in Table 6. The expression of the second generation hCMVmRNA vaccine constructs was validated by Western blot (FIGS. 11A-11E).Further, to test the surface expression of the hCMV pentamer using thesecond generation mRNA vaccine constructs, HeLa cells were transfectedwith 1.25 μg of each of the mRNA vaccine constructs(gH-gL-Ul128-UL130-UL131A at 1:1:1:1:1). The transfected HeLa cells werethen stained with pentamer-specific antibodies and analyzed withFluorescence-activated cell sorting (FACS). The fluorescent cellpopulation indicates surface expression of the hCMV pentamer (FIG. 10).

The second generation hCMV mRNA vaccines encoding the pentamer and gBwere also formulated with Compound 25 lipids and the immunogenicity ofthe formulation was tested (FIGS. 20A-20B). Mice were immunized with atotal dose of 4.2 μg or 1.4 μg of the mRNA vaccine. Mice serum sampleswere taken on day 20 and day 40 post immunization and the serum IgGtiters were assessed on pentamer coated plates or gB coated plates. Thesecond generation hCMV mRNA vaccines induced high levels ofpentamer-specific (FIG. 20A) and gB-specific (FIG. 20B) antibodies.

An HCMV vaccine may comprise, for example, at least one RNApolynucleotide encoded by at least one of the following sequences or byat least one fragment or epitope of the following sequences:

TABLE 6 Second Generation hCMV mRNA Vaccine Construct Sequences Name ofmRNA SEQ Construct Sequence ID NO hCMV_gHTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  58 dimer,AGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCCAGGCC nucleotideTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTC sequenceACGATATGGCGCAGAAGCCGTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAGTGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTCTTCCAAAGCTATAATCAATACTATGTATTCCATATGCCTCGATGTCTCTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAAAGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTCTCGCAGCAGCTAAAGGCACAAGACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCACACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTACTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGACTTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCCTATCAACGCGACAACTTTATACTACGACAAACTGAGAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCTTATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGTTACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTGTTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAAGAATTTATGATCACCTGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCCCTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATCCCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGCCAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGCCTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGTTCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCCCTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCGCTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTAGGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTGGCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATGTACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATGCTTTTGAAGAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACAGTCGTCTCCTCATGATGTCCGTCTACGCGCTATCGGCCATCATCGGCATCTATCTGCTCTACCGCATGCTCAAGACATGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV_gHUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 108 dimer,UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCCAG nucleotideGCCUCCCCUCCUACCUCAUCAUCCUCGCCGUCUGUCUCUUCAGCCACCUACU sequenceUUCGUCACGAUAUGGCGCAGAAGCCGUAUCCGAACCGCUGGACAAAGCGUUUCACCUACUGCUCAACACCUACGGGAGACCCAUCCGCUUCCUGCGUGAAAAUACCACCCAGUGUACCUACAACAGCAGCCUCCGUAACAGCACGGUCGUCAGGGAAAACGCCAUCAGUUUCAACUUCUUCCAAAGCUAUAAUCAAUACUAUGUAUUCCAUAUGCCUCGAUGUCUCUUUGCGGGUCCUCUGGCGGAGCAGUUUCUGAACCAGGUAGAUCUGACCGAAACCCUGGAAAGAUACCAACAGAGACUUAACACUUACGCGCUGGUAUCCAAAGACCUGGCCAGCUACCGAUCUUUCUCGCAGCAGCUAAAGGCACAAGACAGCCUAGGUGAACAGCCCACCACUGUGCCACCGCCCAUUGACCUGUCAAUACCUCACGUUUGGAUGCCACCGCAAACCACUCCACACGGCUGGACAGAAUCACAUACCACCUCAGGACUACACCGACCACACUUUAACCAGACCUGUAUCCUCUUUGAUGGACACGAUCUACUAUUCAGCACCGUCACACCUUGUUUGCACCAAGGCUUUUACCUCAUCGACGAACUACGUUACGUUAAAAUAACACUGACCGAGGACUUCUUCGUAGUUACGGUGUCCAUAGACGACGACACACCCAUGCUGCUUAUCUUCGGCCAUCUUCCACGCGUACUUUUCAAAGCGCCCUAUCAACGCGACAACUUUAUACUACGACAAACUGAGAAACACGAGCUCCUGGUGCUAGUUAAGAAAGAUCAACUGAACCGUCACUCUUAUCUCAAAGACCCGGACUUUCUUGACGCCGCACUUGACUUCAACUACCUAGACCUCAGCGCACUACUACGUAACAGCUUUCACCGUUACGCCGUGGAUGUACUCAAGAGCGGUCGAUGUCAGAUGCUGGACCGCCGCACGGUAGAAAUGGCCUUCGCCUACGCAUUAGCACUGUUCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGUCUCCGUCCCACGGGCCCUAGACCGCCAGGCCGCACUCUUACAAAUACAAGAAUUUAUGAUCACCUGCCUCUCACAAACACCACCACGCACCACGUUGCUGCUGUAUCCCACGGCCGUGGACCUGGCCAAACGAGCCCUUUGGACACCGAAUCAGAUCACCGACAUCACCAGCCUCGUACGCCUGGUCUACAUACUCUCUAAACAGAAUCAGCAACAUCUCAUCCCCCAAUGGGCACUACGACAGAUCGCCGACUUUGCCCUAAAACUACACAAAACGCACCUGGCCUCUUUUCUUUCAGCCUUCGCACGCCAAGAACUCUACCUCAUGGGCAGCCUCGUCCACUCCAUGCUGGUACAUACGACGGAGAGACGCGAAAUCUUCAUCGUAGAAACGGGCCUCUGUUCAUUGGCCGAGCUAUCACACUUUACGCAGUUGUUAGCUCAUCCACACCACGAAUACCUCAGCGACCUGUACACACCCUGUUCCAGUAGCGGGCGACGCGAUCACUCGCUCGAACGCCUCACGCGUCUCUUCCCCGAUGCCACCGUCCCCGCUACCGUUCCCGCCGCCCUCUCCAUCCUAUCUACCAUGCAACCAAGCACGCUGGAAACCUUCCCCGACCUGUUUUGCUUGCCGCUCGGCGAAUCCUUCUCCGCGCUGACCGUCUCCGAACACGUCAGUUAUAUCGUAACAAACCAGUACCUGAUCAAAGGUAUCUCCUACCCUGUCUCCACCACCGUCGUAGGCCAGAGCCUCAUCAUCACCCAGACGGACAGUCAAACUAAAUGCGAACUGACGCGCAACAUGCAUACCACACACAGCAUCACAGUGGCGCUCAACAUUUCGCUAGAAAACUGCGCCUUUUGCCAAAGCGCCCUGCUAGAAUACGACGACACGCAAGGCGUCAUCAACAUCAUGUACAUGCACGACUCGGACGACGUCCUUUUCGCCCUGGAUCCCUACAACGAAGUGGUGGUCUCAUCUCCGCGAACUCACUACCUCAUGCUUUUGAAGAACGGUACGGUACUAGAAGUAACUGACGUCGUCGUGGACGCCACCGACAGUCGUCUCCUCAUGAUGUCCGUCUACGCGCUAUCGGCCAUCAUCGGCAUCUAUCUGCUCUACCGCAUGCUCAAGACAUGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGC hCMV_gHMRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQ  59 dimer,CTYNSSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTET amino acidLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPP sequenceQTTPHGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYIVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSRLLMMSVYALSAIIGIYL LYRMLKTChCMV-gL, TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  60nucleotide AGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTGCCGCCGCC sequenceCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCCTCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAGGTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGTTACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCGGATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGCGATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTCACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAACCGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTCTGCCTGCGTCACCAGCTGGACCCGCCGCTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGCGTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-gL,UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 109 nucleotideUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGUGCCGCC sequenceGCCCGGAUUGCGGCUUCUCUUUCUCACCUGGACCGGUGAUACUGCUGUGGUGUUGCCUUCUGCUGCCCAUUGUUUCCUCAGCCGCCGUCAGCGUCGCUCCUACCGCCGCCGAGAAAGUCCCCGCGGAGUGCCCCGAACUAACGCGCCGAUGCUUGUUGGGUGAGGUGUUUGAGGGUGACAAGUAUGAAAGUUGGCUGCGCCCGUUGGUGAAUGUUACCGGGCGCGAUGGCCCGCUAUCGCAACUUAUCCGUUACCGUCCCGUUACGCCGGAGGCCGCCAACUCCGUGCUGUUGGACGAGGCUUUCCUGGACACUCUGGCCCUGCUGUACAACAAUCCGGAUCAAUUGCGGGCCCUGCUGACGCUGUUGAGCUCGGACACAGCGCCGCGCUGGAUGACGGUGAUGCGCGGCUACAGCGAGUGCGGCGAUGGCUCGCCGGCCGUGUACACGUGCGUGGACGACCUGUGCCGCGGCUACGACCUCACGCGACUGUCAUACGGGCGCAGCAUCUUCACGGAACACGUGUUAGGCUUCGAGCUGGUGCCACCGUCUCUCUUUAACGUGGUGGUGGCCAUACGCAACGAAGCCACGCGUACCAACCGCGCCGUGCGUCUGCCCGUGAGCACCGCUGCCGCGCCCGAGGGCAUCACGCUCUUUUACGGCCUGUACAACGCAGUGAAGGAAUUCUGCCUGCGUCACCAGCUGGACCCGCCGCUGCUACGCCACCUAGAUAAAUACUACGCCGGACUGCCGCCCGAGCUGAAGCAGACGCGCGUCAACCUGCCGGCUCACUCGCGCUAUGGCCCUCAAGCAGUGGAUGCUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUC UGAGUGGGCGGChCMV-gL, MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLG  61amino acid EVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYsequence NNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR hCMV_ULTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  62 128,AGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTCCCAAAG nucleotideATCTGACGCCGTTCTTGACGGCGTTGTGGCTGCTATTGGGTCACAGCCGCGTGC sequenceCGCGGGTGCGCGCAGAAGAATGTTGCGAATTCATAAACGTCAACCACCCGCCGGAACGCTGTTACGATTTCAAAATGTGCAATCGCTTCACCGTCGCGCTGCGGTGTCCGGACGGCGAAGTCTGCTACAGTCCCGAGAAAACGGCTGAGATTCGCGGGATCGTCACCACCATGACCCATTCATTGACACGCCAGGTCGTACACAACAAACTGACGAGCTGCAACTACAATCCGTTATACCTCGAAGCTGACGGGCGAATACGCTGCGGCAAAGTAAACGACAAGGCGCAGTACCTGCTGGGCGCCGCTGGCAGCGTTCCCTATCGATGGATCAATCTGGAATACGACAAGATAACCCGGATCGTGGGCCTGGATCAGTACCTGGAGAGCGTTAAGAAACACAAACGGCTGGATGTGTGCCGCGCTAAAATGGGCTATATGCTGCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV_ULUCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 110 128,UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAGUCCCA nucleotideAAGAUCUGACGCCGUUCUUGACGGCGUUGUGGCUGCUAUUGGGUCACAGCC sequenceGCGUGCCGCGGGUGCGCGCAGAAGAAUGUUGCGAAUUCAUAAACGUCAACCACCCGCCGGAACGCUGUUACGAUUUCAAAAUGUGCAAUCGCUUCACCGUCGCGCUGCGGUGUCCGGACGGCGAAGUCUGCUACAGUCCCGAGAAAACGGCUGAGAUUCGCGGGAUCGUCACCACCAUGACCCAUUCAUUGACACGCCAGGUCGUACACAACAAACUGACGAGCUGCAACUACAAUCCGUUAUACCUCGAAGCUGACGGGCGAAUACGCUGCGGCAAAGUAAACGACAAGGCGCAGUACCUGCUGGGCGCCGCUGGCAGCGUUCCCUAUCGAUGGAUCAAUCUGGAAUACGACAAGAUAACCCGGAUCGUGGGCCUGGAUCAGUACCUGGAGAGCGUUAAGAAACACAAACGGCUGGAUGUGUGCCGCGCUAAAAUGGGCUAUAUGCUGCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC hCMV_ULMSPKDLTPFLTALWLLLGHSRVPRVRAEECCEFINVNHPPERCYDFKMCNRFTVA  63 128, aminoLRCPDGEVCYSPEKTAEIRGIVITMTHSLTRQVVHNKLTSCNYNPLYLEADGRIRC acidGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAK sequence MGYMLQhCMV- TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  64 UL130,AGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCTGCGGCTTC nucleotideTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCCT sequenceGTCTGGCGTCTCCGTGGTCGACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCTTTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACCCTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGTCGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAGATTTTTGGAGCGCACATGGTGCCCAAGCGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATGAAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACTTACACCTTCTGCACCCATCCCAATCTCATCGTTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV-UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 111 UL130,UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCUGCGGC nucleotideUUCUGCUUCGUCACCACUUUCACUGCCUGCUUCUGUGCGCGGUUUGGGCAAC sequenceGCCCUGUCUGGCGUCUCCGUGGUCGACGCUAACAGCAAACCAGAAUCCGUCCCCGCCAUGGUCUAAACUGACGUAUUCCAAACCGCAUGACGCGGCGACGUUUUACUGUCCUUUUCUCUAUCCCUCGCCCCCACGAUCCCCCUUGCAAUUCUCGGGGUUCCAGCGGGUAUCAACGGGUCCCGAGUGUCGCAACGAGACCCUGUAUCUGCUGUACAACCGGGAAGGCCAGACCUUGGUGGAGAGAAGCUCCACCUGGGUGAAAAAGGUGAUCUGGUACCUGAGCGGUCGGAACCAAACCAUCCUCCAACGGAUGCCCCGAACGGCUUCGAAACCGAGCGACGGAAACGUGCAGAUCAGCGUGGAAGACGCCAAGAUUUUUGGAGCGCACAUGGUGCCCAAGCGCUGCUACGCUUCGUCGUCAACGAUGGCACACGUUAUCAGAUGUGUGUGAUGAAGCUGGAGAGCUGGGCUCACGUCUUCCGGGACUACAGCGUGUCUUUUCAGGUGCGAUUGACGUUCACCGAGGCCAAUAACCAGACUUACACCUUCUGCACCCAUCCCAAUCUCAUCGUUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMV-MLRLLLRHHFHCLLLCAVWATPCLASPWSTLTANQNPSPPWSKLTYSKPHDAATF  65 UL130,YCPFLYPSPPRSPLQFSGFQRVSTGPECRNETLYLLYNREGQTLVERSSTWVKKVI amino acidWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDG sequenceTRYQMCVMKLESWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIV hCMVTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  66 UL131A,AGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCGGCTGTGTC nucleotideGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGG sequenceAAACCGCGGAAAAGAACGATTATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGCCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGACCTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGAATCAACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCCGCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGT CTGAGTGGGCGGChCMV UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 112 UL131A,UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCGGCUGU nucleotideGUCGGGUGUGGCUGUCUGUUUGUCUGUGCGCCGUGGUGCUGGGUCAGUGCC sequenceAGCGGGAAACCGCGGAAAAGAACGAUUAUUACCGAGUACCGCAUUACUGGGACGCGUGCUCUCGCGCGCUGCCCGACCAAACCCGUUACAAGUAUGUGGAACAGCUCGUGGACCUCACGUUGAACUACCACUACGAUGCGAGCCACGGCUUGGACAACUUUGACGUGCUCAAGAGAAUCAACGUGACCGAGGUGUCGUUGCUCAUCAGCGACUUUAGACGUCAGAACCGUCGCGGCGGCACCAACAAAAGGACCACGUUCAACGCCGCCGGUUCGCUGGCGCCACACGCCCGGAGCCUCGAGUUCAGCGUGCGGCUCUUUGCCAACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC hCMVMRLCRVWLSVCLCAVVLGQCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYV  67 UL131A,EQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFN amino acidAAGSLAPHARSLEFSVRLFAN sequence hCMV_gB,TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATA  68 nucleotideAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAATCCAGGA sequenceTCTGGTGCCTGGTAGTCTGCGTTAACTTGTGTATCGTCTGTCTGGGTGCTGCGGTTTCCTCATCTTCTACTCGTGGAACTTCTGCTACTCACAGTCACCATTCCTCTCATACGACGTCTGCTGCTCACTCTCGATCCGGTTCAGTCTCTCAACGCGTAACTTCTTCCCAAACGGTCAGCCATGGTGTTAACGAGACCATCTACAACACTACCCTCAAGTACGGAGATGTGGTGGGGGTCAATACCACCAAGTACCCCTATCGCGTGTGTTCTATGGCCCAGGGTACGGATCTTATTCGCTTTGAACGTAATATCGTCTGCACCTCGATGAAGCCCATCAATGAAGACCTGGACGAGGGCATCATGGTGGTCTACAAACGCAACATCGTCGCGCACACCTTTAAGGTACGAGTCTACCAGAAGGTTTTGACGTTTCGTCGTAGCTACGCTTACATCCACACCACTTATCTGCTGGGCAGCAACACGGAATACGTGGCGCCTCCTATGTGGGAGATTCATCATATCAACAGCCACAGTCAGTGCTACAGTTCCTACAGCCGCGTTATAGCAGGCACGGTTTTCGTGGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGACGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCCGCGGCAGCACCTGGCTCTATCGTGAGACCTGTAATCTGAATTGTATGGTGACCATCACTACTGCGCGCTCCAAATATCCTTATCATTTTTTCGCCACTTCCACGGGTGACGTGGTTGACATTTCTCCTTTCTACAACGGAACCAATCGCAATGCCAGCTACTTTGGAGAAAACGCCGACAAGTTTTTCATTTTTCCGAACTACACTATCGTCTCCGACTTTGGAAGACCGAATTCTGCGTTAGAGACCCACAGGTTGGTGGCTTTTCTTGAACGTGCGGACTCGGTGATCTCCTGGGATATACAGGACGAAAAGAATGTCACTTGTCAACTCACTTTCTGGGAAGCCTCGGAACGCACCATTCGTTCCGAAGCCGAGGACTCGTATCACTTTTCTTCTGCCAAAATGACCGCCACTTTCTTATCTAAGAAGCAAGAGGTGAACATGTCCGACTCTGCGCTGGACTGCGTACGTGATGAGGCTATAAATAAGTTACAGCAGATTTTCAATACTTCATACAATCAAACATATGAAAAATATGGAAACGTGTCCGTCTTTGAAACCACTGGTGGTTTGGTAGTGTTCTGGCAAGGTATCAAGCAAAAATCTCTGGTGGAACTCGAACGTTTGGCCAACCGCTCCAGTCTGAATCTTACTCATAATAGAACCAAAAGAAGTACAGATGGCAACAATGCAACTCATTTATCCAACATGGAATCGGTGCACAATCTGGTCTACGCCCAGCTGCAGTTCACCTATGACACGTTGCGCGGTTACATCAACCGGGCGCTGGCGCAAATCGCAGAAGCCTGGTGTGTGGATCAACGGCGCACCCTAGAGGTCTTCAAGGAACTCAGCAAGATCAACCCGTCAGCCATTCTCTCGGCCATTTACAACAAACCGATTGCCGCGCGTTTCATGGGTGATGTCTTGGGCCTGGCCAGCTGCGTGACCATCAACCAAACCAGCGTCAAGGTGCTGCGTGATATGAACGTGAAGGAGTCGCCAGGACGCTGCTACTCACGACCCGTGGTCATCTTTAATTTCGCCAACAGCTCGTACGTGCAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAGCAGTATCTCCACCGTCGACAGCATGATCGCCCTGGATATCGACCCGCTGGAAAATACCGACTTCAGGGTACTGGAACTTTACTCGCAGAAAGAGCTGCGTTCCAGCAACGTTTTTGACCTCGAAGAGATCATGCGCGAATTCAACTCGTACAAGCAGCGGGTAAAGTACGTGGAGGACAAGGTAGTCGACCCGCTACCGCCCTACCTCAAGGGTCTGGACGACCTCATGAGCGGCCTGGGCGCCGCGGGAAAGGCCGTTGGCGTAGCCATTGGGGCCGTGGGTGGCGCGGTGGCCTCCGTGGTCGAAGGCGTTGCCACCTTCCTCAAAAACCCCTTCGGAGCGTTCACCATCATCCTCGTGGCCATAGCTGTAGTCATTATCACTTATTTGATCTATACTCGACAGCGGCGTTTGTGCACGCAGCCGCTGCAGAACCTCTTTCCCTATCTGGTGTCCGCCGACGGGACCACCGTGACGTCGGGCAGCACCAAAGACACGTCGTTACAGGCTCCGCCTTCCTACGAGGAAAGTGTTTATAATTCTGGTCGCAAAGGACCGGGACCACCGTCGTCTGATGCATCCACGGCGGCTCCGCCTTACACCAACGAGCAGGCTTACCAGATGCTTCTGGCCCTGGCCCGTCTGGACGCAGAGCAGCGAGCGCAGCAGAACGGTACAGATTCTTTGGACGGACGGACTGGCACGCAGGACAAGGGACAGAAGCCCAACCTACTAGACCGACTGCGACATCGCAAAAACGGCTACCGACACTTGAAAGACTCTGACGAAGAAGAGAACGTCTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hCMV_gB,UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA 113 nucleotideUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAUCCA sequenceGGAUCUGGUGCCUGGUAGUCUGCGUUAACUUGUGUAUCGUCUGUCUGGGUGCUGCGGUUUCCUCAUCUUCUACUCGUGGAACUUCUGCUACUCACAGUCACCAUUCCUCUCAUACGACGUCUGCUGCUCACUCUCGAUCCGGUUCAGUCUCUCAACGCGUAACUUCUUCCCAAACGGUCAGCCAUGGUGUUAACGAGACCAUCUACAACACUACCCUCAAGUACGGAGAUGUGGUGGGGGUCAAUACCACCAAGUACCCCUAUCGCGUGUGUUCUAUGGCCCAGGGUACGGAUCUUAUUCGCUUUGAACGUAAUAUCGUCUGCACCUCGAUGAAGCCCAUCAAUGAAGACCUGGACGAGGGCAUCAUGGUGGUCUACAAACGCAACAUCGUCGCGCACACCUUUAAGGUACGAGUCUACCAGAAGGUUUUGACGUUUCGUCGUAGCUACGCUUACAUCCACACCACUUAUCUGCUGGGCAGCAACACGGAAUACGUGGCGCCUCCUAUGUGGGAGAUUCAUCAUAUCAACAGCCACAGUCAGUGCUACAGUUCCUACAGCCGCGUUAUAGCAGGCACGGUUUUCGUGGCUUAUCAUAGGGACAGCUAUGAAAACAAAACCAUGCAAUUAAUGCCCGACGAUUAUUCCAACACCCACAGUACCCGUUACGUGACGGUCAAGGAUCAAUGGCACAGCCGCGGCAGCACCUGGCUCUAUCGUGAGACCUGUAAUCUGAAUUGUAUGGUGACCAUCACUACUGCGCGCUCCAAAUAUCCUUAUCAUUUUUUCGCCACUUCCACGGGUGACGUGGUUGACAUUUCUCCUUUCUACAACGGAACCAAUCGCAAUGCCAGCUACUUUGGAGAAAACGCCGACAAGUUUUUCAUUUUUCCGAACUACACUAUCGUCUCCGACUUUGGAAGACCGAAUUCUGCGUUAGAGACCCACAGGUUGGUGGCUUUUCUUGAACGUGCGGACUCGGUGAUCUCCUGGGAUAUACAGGACGAAAAGAAUGUCACUUGUCAACUCACUUUCUGGGAAGCCUCGGAACGCACCAUUCGUUCCGAAGCCGAGGACUCGUAUCACUUUUCUUCUGCCAAAAUGACCGCCACUUUCUUAUCUAAGAAGCAAGAGGUGAACAUGUCCGACUCUGCGCUGGACUGCGUACGUGAUGAGGCUAUAAAUAAGUUACAGCAGAUUUUCAAUACUUCAUACAAUCAAACAUAUGAAAAAUAUGGAAACGUGUCCGUCUUUGAAACCACUGGUGGUUUGGUAGUGUUCUGGCAAGGUAUCAAGCAAAAAUCUCUGGUGGAACUCGAACGUUUGGCCAACCGCUCCAGUCUGAAUCUUACUCAUAAUAGAACCAAAAGAAGUACAGAUGGCAACAAUGCAACUCAUUUAUCCAACAUGGAAUCGGUGCACAAUCUGGUCUACGCCCAGCUGCAGUUCACCUAUGACACGUUGCGCGGUUACAUCAACCGGGCGCUGGCGCAAAUCGCAGAAGCCUGGUGUGUGGAUCAACGGCGCACCCUAGAGGUCUUCAAGGAACUCAGCAAGAUCAACCCGUCAGCCAUUCUCUCGGCCAUUUACAACAAACCGAUUGCCGCGCGUUUCAUGGGUGAUGUCUUGGGCCUGGCCAGCUGCGUGACCAUCAACCAAACCAGCGUCAAGGUGCUGCGUGAUAUGAACGUGAAGGAGUCGCCAGGACGCUGCUACUCACGACCCGUGGUCAUCUUUAAUUUCGCCAACAGCUCGUACGUGCAGUACGGUCAACUGGGCGAGGACAACGAAAUCCUGUUGGGCAACCACCGCACUGAGGAAUGUCAGCUUCCCAGCCUCAAGAUCUUCAUCGCCGGGAACUCGGCCUACGAGUACGUGGACUACCUCUUCAAACGCAUGAUUGACCUCAGCAGUAUCUCCACCGUCGACAGCAUGAUCGCCCUGGAUAUCGACCCGCUGGAAAAUACCGACUUCAGGGUACUGGAACUUUACUCGCAGAAAGAGCUGCGUUCCAGCAACGUUUUUGACCUCGAAGAGAUCAUGCGCGAAUUCAACUCGUACAAGCAGCGGGUAAAGUACGUGGAGGACAAGGUAGUCGACCCGCUACCGCCCUACCUCAAGGGUCUGGACGACCUCAUGAGCGGCCUGGGCGCCGCGGGAAAGGCCGUUGGCGUAGCCAUUGGGGCCGUGGGUGGCGCGGUGGCCUCCGUGGUCGAAGGCGUUGCCACCUUCCUCAAAAACCCCUUCGGAGCGUUCACCAUCAUCCUCGUGGCCAUAGCUGUAGUCAUUAUCACUUAUUUGAUCUAUACUCGACAGCGGCGUUUGUGCACGCAGCCGCUGCAGAACCUCUUUCCCUAUCUGGUGUCCGCCGACGGGACCACCGUGACGUCGGGCAGCACCAAAGACACGUCGUUACAGGCUCCGCCUUCCUACGAGGAAAGUGUUUAUAAUUCUGGUCGCAAAGGACCGGGACCACCGUCGUCUGAUGCAUCCACGGCGGCUCCGCCUUACACCAACGAGCAGGCUUACCAGAUGCUUCUGGCCCUGGCCCGUCUGGACGCAGAGCAGCGAGCGCAGCAGAACGGUACAGAUUCUUUGGACGGACGGACUGGCACGCAGGACAAGGGACAGAAGCCCAACCUACUAGACCGACUGCGACAUCGCAAAAACGGCUACCGACACUUGAAAGACUCUGACGAAGAAGAGAACGUCUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC hCMV_gB,MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQR  69 amino acidVTSSQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVC sequenceTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIITYLIYTRQRRLCTQPLQNLFPYLVSADGTTVTSGSTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRAQQNGTDSLDGRTGTQDKGQKPNLLDRLRHRK NGYRHLKDSDEEENV

Example 26: 2A Peptide Linked Pentameric Subunits

Multivalent mRNA vaccine constructs encoding the subunits of the hCMVpentamer (gH, gL, UL128, UL130, and U1131A) were designed. Themultivalent mRNA encoded pentamer subunits were linked with 2Aself-cleaving peptides (FIG. 15), which allows the linked subunits toprocess into individual subunits. 1 μg of the mRNA vaccine constructsencoding a 2A peptide linked gH-gL were transfected into 293T cells. Thecells were harvested 24 hours post transfection and the cleavage of the2A peptide were analyzed by detecting individual gH or gL subunits usingWestern blotting. Individual gH and gL were detected, indicatingsuccessful expression of the construct and cleavage of the 2A peptide(FIG. 16). Further, processed gH or gL when expressed in HeLa cells,dimerized, and translocated to the cell surface 24 hours after the Helacells were transfected with 0.5 μg of mRNA encoding the 2A linked gH-gL(FIG. 17).

Example 27: Comparison of Equimolar Vs Equal Mass of Pentamer

Pentameric formulations containing the pentameric subunit mRNAs atequimolar concentrations were compared to pentameric formulationscontaining the pentameric subunit mRNAs in equal mass. FIG. 18demonstrates high and sustained titers of anti-pentamer binding andneutralizing antibodies in mice. FIG. 18A depicts a graph showinganti-pentamer antibody titers. Equimolar and equal mass formulations ofthe pentameric mRNAs were found to be equally effective. FIG. 18Bdepicts a graph showing neutralizing titers measured on ARPE19epithelial cells infected with hCMV strain VR1814. Equimolar and equalmass formulations of the pentameric mRNAs were compared and were foundto be equally effective. Neutralizing titers were found to beapproximately 25 fold higher than CytoGam®.

Example 28: Neutralization Activity is Dependent on Anti-PentamerAntibodies

Neutralization data was assessed and compared against CytoGam®. FIG. 19demonstrates that neutralization activity against epithelial cellinfection is dependent on anti-pentamer antibodies. FIG. 19A shows thatthe depleting protein was either the pentamer or a gH/gL dimer. FIG. 19Band FIG. 19C depict graphs showing neutralization. FIG. 19B showsneutralization by sera from mice immunized with the pentamer or with agH/gL dimer. FIG. 19C shows neutralization by CytoGam® combined with thepentamer or with a gH/gL.

Example 28:Phase 1 Clinical Trial for Prevention of Mother-to-Child(Congenital) Transmission

A phase 1 clinical trial is conducted to assess the safety of thechemically modified or unmodified hCMV mRNA vaccine encoding thepentameric complex (gH, gL, UL128, UL130, and UL131A)+gB in humans andto evaluate the ability of the hCMV mRNA vaccines to induce an immuneresponse. One hundred and twenty (120) volunteers (both females andmales) between ages 18-49 are enrolled in the clinical trial. Thevolunteers are tested for CMV prior to the start of the clinical trial.Sixty (60) of the healthy volunteers are CMV⁺, while the other sixty(60) are CMV⁻.

The healthy volunteers are divided into three dosage groups, each dosagegroup receiving a different dose of the hCMV mRNA vaccine (e.g., low,medium, or high). For each dosage group (n=40), the hCMV mRNA vaccine isadministered intramuscularly (IM, n=20) or intravenously (IV, n=20).Thus, the 120 volunteers are placed into 6 groups (referred to as a“dose arm”): low dose-IM (n=20), low dose-IV (n=20), medium dose-IM(n=20), medium dose-IV (n=20), high dose-IM (n=20), high dose-IV (n=20).In each dose arm, the volunteers are separated into two cohorts: thesafety cohort (n=4, 2 receiving vaccines and 2 receiving placebos); andthe expansion cohort (n=16, 13 receiving vaccines and 3 receivingplacebos). The immunization of the volunteers in the expansion cohortstarts 7 days after the last healthy volunteer in the safety cohort hasbeen immunized.

hCMV vaccines or placebos are given to the volunteers in the 6 dose armson day 1, day 31, and day 61. It is a double blind clinical trial. Thevolunteers are followed up to a year. Blood samples are taken on day 1,day 8, day 22, day 30, day 44, 6 months, and 1 year after the firstimmunization.

Neutralizing hCMV antibody titers in the blood samples are measuredusing an Enzyme-linked ImmunoSpot (ELISPOT) assay or using a lowcytometric intracellular cytokine staining (ICS) assay. Sustainedneutralization antibody titers and strong anamnestic responses areexpected in volunteers who received the hCMV mRNA vaccines by 12 months.The level of IgG induced by the hCMV mRNA vaccines are expected to be atleast 4 times above the baseline (a clinical endpoint). Theneutralization antibody titer in the blood samples of volunteers whoreceived the hCMV mRNA vaccine, measured in a plaque reductionneutralization test (PRNT50) in both epithelial and fibroblast cells, isexpected to be higher than that of Cytogam® (a clinical end point).Early signal of efficacy (ESOE) can also be indicated by measuring theviral load in urine and saliva of the volunteers by PCR on day 1, 6months, and 12 months.

Parameters indicating safety of the vaccine are measured. Immunizedvolunteers are evaluated for clinical signs of hCMV infection (aclinical endpoint). Biochemical assays are performed to assess thecoagulation parameters and the blood level of C-reactive proteins (CRP).The hCMV mRNA vaccine is expected to be safe.

Once safety and immunogenicity have been demonstrated, trials areconducted among target populations in phase 2 clinical trials. In someembodiments, suitable dose levels chosen from phase 1 trials will beused in phase 2 trials.

Example 29: Phase 2 Clinical Trial—Day Care Personnel

A phase 2 clinical trial is conducted to evaluate the chemicallymodified or unmodified hCMV mRNA vaccine encoding the pentameric complex(gH, gL, UL128, UL130, and UL131A)+gB in humans in the followingpopulations: seronegative and seropositive (safety cohort) day carepersonnel; seronegative and seropositive (safety cohort) parents whohave a child in daycare; and seronegative toddlers.

Three hundred (300) subjects are enrolled in the phase 2 clinical trialand are grouped as in the phase 1 clinical trial described in Example26. All subjects are immunized with the same dose of hCMV mRNA vaccine.In some embodiments a dose-response trial is conducted. Subjects receivethe first dose of the vaccine on day 1, which is 2-4 weeks prior to theinitiation of immunosuppressive therapy, and receive boosts atapproximately 1, 3, and 6 months. It is a double blind clinical trial.The subjects are followed up to a year. Blood samples are taken onapproximately day 1, day 8, day 22, day 30, day 44, 6 months, and 1 yearafter the first immunization.

The safety and immunogenicity of the vaccines are assessed using methodsdescribed in the phase 1 trial described in Example 28. A vaccineefficacy of at least 70% is expected. Clinical endpoints for this trialinclude infection in urine and/or saliva, as detected by PCR. The hCMVmRNA vaccine is expected to induce immune response and generateneutralizing antibodies. The safety profile is also expected to be high.

Example 30: Phase 3 Clinical Trial—Adolescent Boys and Girls

A phase 3 clinical trial is conducted in adolescent boys and girls.Optionally, a phase 3 clinical trial is also conducted in toddlers. NoCMV screening is performed prior to enrollment.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

1-104. (canceled)
 105. A human cytomegalovirus (hCMV) immunogeniccomposition comprising at least one ribonucleic acid (RNA)polynucleotide having an open reading frame encoding at least one hCMVantigenic polypeptide formulated in a lipid nanoparticle comprising anionizable cationic lipid of Compound 25:


106. The hCMV immunogenic composition of claim 105, wherein the lipidnanoparticle further comprises a non-cationic lipid, a sterol, and aPEG-modified lipid.
 107. The hCMV immunogenic composition of claim 106,wherein the lipid nanoparticle comprises a molar ratio of: 20-60%ionizable cationic lipid of Compound 25, 0.5-15% PEG-modified lipid,25-55% sterol, and 5-25% non-cationic lipid.
 108. The hCMV immunogeniccomposition of claim 107, wherein the lipid nanoparticle comprises amolar ratio of 40%-50% ionizable cationic lipid of Compound
 25. 109. ThehCMV immunogenic composition of claim 108, wherein the lipidnanoparticle comprises a molar ratio of 50% ionizable cationic lipid ofCompound
 25. 110. The hCMV immunogenic composition of claim 108, whereinthe lipid nanoparticle comprises a molar ratio of 49% ionizable cationiclipid of Compound
 25. 111. The hCMV immunogenic composition of claim108, wherein the lipid nanoparticle comprises a molar ratio of 48%ionizable cationic lipid of Compound
 25. 112. The hCMV immunogeniccomposition of claim 108, wherein the lipid nanoparticle comprises amolar ratio of 47% ionizable cationic lipid of Compound
 25. 113. ThehCMV immunogenic composition of claim 108, wherein the lipidnanoparticle comprises a molar ratio of 46% ionizable cationic lipid ofCompound
 25. 114. The hCMV immunogenic composition of claim 108, whereinthe lipid nanoparticle comprises a molar ratio of 45% ionizable cationiclipid of Compound
 25. 115. The hCMV immunogenic composition of claim106, wherein the non-cationic lipid is a neutral lipid and the sterol ischolesterol.
 116. The hCMV immunogenic composition of claim 105, whereinthe at least one hCMV antigenic polypeptide is selected from hCMV gBprotein, hCMV gH protein, hCMV gL protein, hCMV UL128 protein, hCMVUL130 protein, hCMV UL131A protein, and combinations thereof.
 117. ThehCMV immunogenic composition of claim 116, wherein the at least one hCMVantigenic polypeptide includes a hCMV gB protein, a hCMV gH protein, ahCMV gL protein, a hCMV UL128 protein, a hCMV UL130 protein, and a hCMVUL131A protein.
 118. The hCMV immunogenic composition of claim 105,wherein the at least one RNA polynucleotide comprises at least onechemical modification.
 119. The hCMV immunogenic composition of claim118, wherein the chemical modification is selected from pseudouridine,1-methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.
 120. ThehCMV immunogenic composition of claim 119, wherein the chemicalmodification is a 1-methylpseudouridine or 1-ethylpseudouridine. 121.The hCMV immunogenic composition of claim 118, wherein the chemicalmodification is in the carbon 5-position of the uracil.
 122. The hCMVimmunogenic composition of claim 105, wherein the RNA polynucleotidefurther includes a 5′ terminal cap.
 123. The hCMV immunogeniccomposition of claim 122, wherein the 5′ terminal cap is7mG(5′)ppp(5′)NlmpNp.
 124. The hCMV immunogenic composition of claim105, wherein the open reading frame is codon-optimized.
 125. The hCMVimmunogenic composition of claim 105, wherein the at least one RNApolynucleotide is a messenger RNA (mRNA) polynucleotide.
 126. A methodof inducing an antigen-specific immune response in a subject, the methodcomprising administering to the subject the immunogenic composition ofclaim 105 in an amount effective to produce an antigen-specific immuneresponse in the subject.
 127. A method of preventing congenital hCMVinfection comprising administering to a woman of child-bearing age atherapeutically effective amount of a human cytomegalovirus (hCMV)immunogenic composition comprising: i) at least one RNA polynucleotidehaving one or more open reading frames encoding HCMV antigenicpolypeptides gH, gL, UL128, UL130, and/or UL131A; ii) an RNApolynucleotide having an open reading frame encoding hCMV antigenicpolypeptide gB; and iii) a pharmaceutically acceptable carrier orexcipient.
 128. The method of claim 127, wherein the hCMV immunogeniccomposition comprises: (a) at least one RNA polynucleotide comprising anopen reading frame encoding a hCMV gH polypeptide; (b) at least one RNApolynucleotide comprising an open reading frame encoding a hCMV gLpolypeptide; (c) at least one RNA polynucleotide comprising an openreading frame encoding a hCMV UL128 polypeptide; (d) at least one RNApolynucleotide comprising an open reading frame encoding a hCMV UL130polypeptide; (e) at least one RNA polynucleotide comprising an openreading frame encoding a hCMV UL131A polypeptide; and (f) at least oneRNA polynucleotide comprising an open reading frame encoding a hCMV gBpolypeptide.
 129. The method of claim 128, wherein the hCMV gHpolypeptide comprises an amino acid sequence that has at least 90%identity to the amino acid sequence of SEQ ID NO: 59, the hCMV gLpolypeptide comprises an amino acid sequence that has at least 90%identity to the amino acid sequence of SEQ ID NO: 61, the hCMV UL128polypeptide comprises an amino acid sequence that has at least 90%identity to the amino acid sequence of SEQ ID NO: 63, the hCMV UL130polypeptide comprises an amino acid sequence that has at least 90%identity to the amino acid sequence of SEQ ID NO: 65, the hCMV UL131Apolypeptide comprises an amino acid sequence that has at least 90%identity to the amino acid sequence of SEQ ID NO: 67, and/or the hCMV gBprotein comprises an amino acid sequence that has at least 90% identityto the amino acid sequence of SEQ ID NO:
 69. 130. The method of claim129, wherein the hCMV gH polypeptide comprises the amino acid sequenceof SEQ ID NO: 59, the hCMV gL polypeptide comprises the amino acidsequence of SEQ ID NO: 61, the hCMV UL128 polypeptide comprises theamino acid sequence of SEQ ID NO: 63, the hCMV UL130 polypeptidecomprises the amino acid sequence of SEQ ID NO: 65, the hCMV UL131Apolypeptide comprises the amino acid sequence of SEQ ID NO: 67, and/orthe hCMV gB protein comprises the amino acid sequence of SEQ ID NO: 69.131. The method of claim 128, wherein the RNA polynucleotide of (a)comprises a sequence that has at least 90% identity to the nucleotidesequence of SEQ ID NO: 108, the RNA polynucleotide of (b) comprises asequence that has at least 90% identity to the nucleotide sequence ofSEQ ID NO: 109, the RNA polynucleotide of (c) comprises a sequence thathas at least 90% identity to the nucleotide sequence of SEQ ID NO: 110,the RNA polynucleotide of (d) comprises a sequence that has at least 90%identity to the nucleotide sequence of SEQ ID NO: 93, the RNApolynucleotide of (e) comprises a sequence that has at least 90%identity to the nucleotide sequence of SEQ ID NO: 112, and/or the RNApolynucleotide of (f) comprises a sequence that has at least 90%identity to the nucleotide sequence of SEQ ID NO:
 83. 132. The method ofclaim 128, wherein the hCMV vaccine comprises (a) a RNA polynucleotidecomprising nucleotides 46-2437 of SEQ ID NO: 108, (b) a RNApolynucleotide comprising nucleotides 46-1045 of SEQ ID NO: 109, (c) aRNA polynucleotide comprising nucleotides 46-724 of SEQ ID NO: 110, (d)a RNA polynucleotide comprising nucleotides 46-853 of SEQ ID NO: 93, (e)a RNA polynucleotide comprising nucleotides 46-598 of SEQ ID NO: 112,and (f) a RNA polynucleotide comprising nucleotides 46-2932 of SEQ IDNO:
 83. 133. The method of claim 127, wherein the RNA polynucleotidesare messenger RNA (mRNA) polynucleotides.
 134. The method of claim 133,wherein the mRNA polynucleotides further comprise polyA tails.